Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab

ABSTRACT

The present application describes uses for Pertuzumab, a first-in-class HER2 dimerization inhibitor. In particular, the application describes methods for extending progression free survival in a HER2-positive breast cancer patient population; and combining two HER2 antibodies to treat HER2-positive cancer without increasing cardiac toxicity.

This is a continuation application of U.S. application Ser. No.16/123,809, filed on Sep. 6, 2018, which is a continuation applicationof U.S. application Ser. No. 15/058,520, filed on Mar. 2, 2016 (nowabandoned), which is a continuation application of U.S. application Ser.No. 13/649,591, filed on Oct. 11, 2012 (now abandoned), which claims thebenefit under 35 USC § 119(e) of U.S. Provisional Application Ser. No.61/547,535, filed on Oct. 14, 2011. U.S. Provisional Application Ser.No. 61/567,015, filed on Dec. 5, 2011, U.S. Provisional Application Ser.No. 61/657,669, filed on Jun. 8, 2012. U.S. Provisional Application Ser.No. 61/682,037, filed on Aug. 10, 2012 and U.S. Provisional ApplicationSer. No. 61/694,584, filed on Aug. 29, 2012, which are incorporated byreference in entirety.

FIELD OF THE INVENTION

The present invention concerns uses for and articles of manufactureincluding Pertuzumab, a first-in-class HER2 dimerization inhibitor.

In particular, the invention concerns extending progression freesurvival in a HER2-positive breast cancer patient population; combiningtwo HER2 antibodies to treat HER2-positive cancer without increasingcardiac toxicity; treating early-stage HER2-positive breast cancer,treating HER2-positive cancer by co-administering a mixture ofPertuzumab and Trastuzumab from the same intravenous bag; treatingHER2-positive metastatic gastric cancer; treating HER2-positive breastcancer with Pertuzumab, Trastuzumab and Vinorelbine; treatingHER2-positive breast cancer with Pertuzumab, Trastuzumab and aromataseinhibitor; and treating low HER3 ovarian, primary peritoneal, orfallopian tube cancer.

It also concerns an article of manufacture comprising a vial withPertuzumab therein and a package insert providing safety and/or efficacydata thereon; a method of making the article of manufacture; and amethod of ensuring safe and effective use of Pertuzumab related thereto.

In addition the invention concerns an intravenous (IV) bag containing astable mixture of Pertuzumab and Trastuzumab suitable for administrationto a cancer patient.

BACKGROUND OF THE INVENTION

Members of the HER family of receptor tyrosine kinases are importantmediators of cell growth, differentiation and survival. The receptorfamily includes four distinct members including epidermal growth factorreceptor (EGFR, ErbB1, or HER1). HER2 (ErbB2 or p185^(neu)), HER3(ErbB3) and HER4 (ErbB4 or tyro2). Members of the receptor family havebeen implicated in various types of human malignancy.

A recombinant humanized version of the murine anti-HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2. Trastuzumab or HERCEPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anti-cancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)).

Trastuzumab received marketing approval from the Food and DrugAdministration Sep. 25, 1998 for the treatment of patients withmetastatic breast cancer whose tumors overexpress the HER2 protein. Atpresent, Trastuzumab is approved for use as a single agent or incombination with chemotherapy or hormone therapy in the metastaticsetting, and as single agent or in combination with chemotherapy asadjuvant treatment for patients with early-stage HER2-positive breastcancer. Trastuzumab-based therapy is now the recommended treatment forpatients with HER2-positive early-stage breast cancer who do not havecontraindications for its use (Herceptin® prescribing information; NCCNGuidelines, version 2.2011). Trastuzumab plus Docetaxel (or paclitaxel)is a registered standard of care in the first-line metastatic breastcancer (MBC) treatment setting (Slamon et al. N Engl J Med. 2001;344(11):783-792.; Marty et al. J Clin Oncol. 2005; 23(19):4265-4274).

While the administration of Trastuzumab has led to excellent results inthe treatment of breast cancer, recent data from a clinical trial oflapatinib appear to suggest that even with administration ofTrastuzumab, HER2 plays an active role in tumor biology (Geyer et al., NEngl J Med 2006; 355:2733-2743).

Patients treated with the HER2 antibody Trastuzumab are selected fortherapy based on HER2 expression. See, for example, WO99/31140 (Paton etal.), US2003/0170234A1 (Hellmann, S.), and US2003/0147884 (Paton etal.); as well as WO01/89566. US2002/0064785, and US2003/0134344 (Mass etal.). See, also, U.S. Pat. Nos. 6,573,043, 6,905,830, andUS2003/0152987, Cohen et al., concerning immunohistochemistry (IHC) andfluorescence in situ hybridization (FISH) for detecting HER2overexpression and amplification. Thus, the optimal management ofmetastatic breast cancer now takes into account not only a patient'sgeneral condition, medical history, and receptor status, but also theHER2 status.

Pertuzumab (also known as recombinant humanized monoclonal antibody 2C4(rhuMAb 2C4); Genentech, Inc, South San Francisco) represents the firstin a new class of agents known as HER dimerization inhibitors (HDI) andfunctions to inhibit the ability of HER2 to form active heterodimers orhomodimers with other HER receptors (such as EGFR/HER1, HER2, HER3 andHER4). See, for example, Harari and Yarden Oncogene 19:6102-14 (2000);Yarden and Sliwkowski. Nat Rev Mol Cell Biol 2:127-37 (2001); SliwkowskiNat Struct Biol 10:158-9 (2003); Cho et al. Nature 421:756-60 (2003);and Malik et al. Pro Am Soc Cancer Res 44:176-7 (2003).

Pertuzumab blockade of the formation of HER2-HER3 heterodimers in tumorcells has been demonstrated to inhibit critical cell signaling, whichresults in reduced tumor proliferation and survival (Agus et al. CancerCell 2:127-37 (2002)).

Pertuzumab has undergone testing as a single agent in the clinic with aphase Ia trial in patients with advanced cancers and phase II trials inpatients with ovarian cancer and breast cancer as well as lung andprostate cancer. In a Phase I study, patients with incurable, locallyadvanced, recurrent or metastatic solid tumors that had progressedduring or after standard therapy were treated with Pertuzumab givenintravenously every 3 weeks. Pertuzumab was generally well tolerated.Tumor regression was achieved in 3 of 20 patients evaluable forresponse. Two patients had confirmed partial responses. Stable diseaselasting for more than 2.5 months was observed in 6 of 21 patients (Aguset al. Pro Am Soc Clin Oncol 22:192 (2003)). At doses of 2.0-15 mg/kg,the pharmacokinetics of Pertuzumab was linear, and mean clearance rangedfrom 2.69 to 3.74 mL/day/kg and the mean terminal elimination half-liferanged from 15.3 to 27.6 days. Antibodies to Pertuzumab were notdetected (Allison et al. Pro Am Soc Clin Oncol 22:197 (2003)).

US 2006/0034842 describes methods for treating ErbB-expressing cancerwith anti-ErbB2 antibody combinations. US 2008/0102069 describes the useof Trastuzumab and Pertuzumab in the treatment of HER2-positivemetastatic cancer, such as breast cancer. Baselga et al., J Clin Oncol,2007 ASCO Annual Meeting Proceedings Part I, Col. 25, No. 18S (June 20Supplement), 2007:1004 report the treatment of patients with pre-treatedHER2-positive breast cancer, which has progressed during treatment withTrastuzumab, with a combination of Trastuzumab and Pertuzumab. Porteraet al., J Clin Oncol. 2007 ASCO Annual Meeting Proceedings Part 1. Vol.25, No. 18S (June 20 Supplement), 2007:1028 evaluated the efficacy andsafety of Trastuzumab+Pertuzumab combination therapy in HER2-positivebreast cancer patients, who had progressive disease on Trastuzumab-basedtherapy. The authors concluded that further evaluation of the efficacyof combination treatment was required to define the overall risk andbenefit of this treatment regimen.

Pertuzumab has been evaluated in Phase II studies in combination withTrastuzumab in patients with HER2-positive metastatic breast cancer whohave previously received Trastuzumab for metastatic disease. One study,conducted by the National cancer Institute (NCI), enrolled 11 patientswith previously treated HER2-positive metastatic breast cancer. Two outof the 11 patients exhibited a partial response (PR) (Baselga et al., JClin Oncol 2007 ASCO Annual Meeting Proceedings; 25:18S (June 20Supplement); 1004. The results of a Phase II neoadjuvant studyevaluating the effect of a novel combination regimen of Pertuzumab andTrastuzumab plus chemotherapy (Docetaxel) in women with early-stageHER2-positive breast cancer, presented at the CTRC-AACR San AntonioBreast Cancer Symposium (SABCS), Dec. 8-12, 2010, showed that the twoHER2 antibodies plus Docetaxel given in the neoadjuvant setting prior tosurgery significantly improved the rate of complete tumor disappearance(pathological complete response rate, pCR, of 45.8 percent) in thebreast by more than half compared to Trastuzumab plus Docetaxel (pCR of29.0 percent), p=0.014.

Patent Publications related to HER2 antibodies include: U.S. Pat. Nos.5,677,171; 5,720,937; 5,720,954; 5,725,856; 5,770,195; 5,772,997;6,165,464; 6,387,371; 6,399,063; 6,015,567; 6,333,169; 4,968,603;5,821,337; 6,054,297; 6,407,213; 6,639,055; 6,719,971; 6,800,738;8,075,890; 5,648,237; 7,018,809; 6,267,958; 6,685,940; 6,821,515;7,060,268; 7,682,609; 7,371,376; 6,127,526; 6,333,398; 6,797,814;6,339,142; 6,417,335; 6,489,447; 7,074,404; 7,531,645; 7,846,441;7,892,549; 8,075,892; 6,573,043; 6,905,830; 7,129,051; 7,344,840;7,468,252; 7,674,589; 7,919,254; 6,949,245; 7,485,302; 7,498,030;7,501,122; 7,537,931; 7,618,631; 7,862,817; 7,041,292; 6,627,196;7,371,379; 6,632,979; 7,097,840; 7,575,748; 6,984,494; 7,279,287;7,811,773; 7,993,834; 8,076,066; 8,044,017; 7,435,797; 7,850,966;7,485,704; 7,807,799; 8,142,784; 7,560,111; 7,879,325; 8,241,630;7,449,184; 8,163,287; 7,700,299; 7,981,418; 8,247,397; and US2010/0016556; US 2005/0244929; US 2001/0014326; US 2003/0202972; US2006/0099201; US 2010/0158899; US 2011/0236383; US 2011/0033460; US2008/0286280; US 2005/0063972; US 2006/0182739; US 2009/0220492; US2003/0147884; US 2004/0037823; US 2005/0002928; US 2007/0292419; US2008/0187533; US 2011/0250194; US 2012/0034213; US 2003/0152987; US2005/0100944 US 2006/0183150; US 2008/0050748; US 2009/0155803; US2010/0120053; US 2005/0244417; US 2007/0026001; US 2008/0160026; US2008/0241146; US 2005/0208043; US 2005/0238640; US 2006/0034842; US2006/0073143; US 2006/0193854; US 2006/0198843; US 2011/0129464; US2007/0184055; US 2007/0269429; US 2008/0050373; US 2006/0083739; US2009/0087432; US 2006/0210561; US 2002/0035736; US 2002/0001587; US2008/0226659; US 2002/0090662; US 2006/0046270; US 2008/0108096; US2007/0166753; US 2008/0112958; US 2009/0239236; US 2012/0034609; US2012/0093838; US 2004/0082047; US 2012/0065381; US 2009/0187007; US2011/0159014; US 2004/0106161; US 2011/0117096; US 2004/0258685; US2009/0148402; US 2009/0099344; US 2006/0034840; US 2011/0064737; US2005/0276812; US 2008/0171040; US 2009/0202536; US 2006/0013819; US2012/0107391; US 2006/0018899; US 2009/0285837; US 2011/0117097; US2006/0088523; US 2010/0015157; US 2006/0121044; US 2008/0317753; US2006/0165702; US 2009/0081223; US 2006/0188509; US 2009/0155259; US2011/0165157; US 2006/0204505; US 2006/0212956; US 2006/0275305; US2012/0003217; US 2007/0009976 US 2007/0020261; US 2007/0037228; US2010/0112603; US 2006/0067930; US 2007/0224203; US 2011/0064736; US2008/0038271; US 2008/0050385; US 2010/0285010; US 2011/0223159; US2008/0102069; US 2010/0008975; US 2011/0245103; US 2011/0246399; US2011/0027190; US 2010/0298156; US 2011/0151454; US 2011/0223619; US2012/0107302; US 2009/0098135; US 2009/0148435; US 2009/0202546; US2009/0226455; US 2009/0317387; US 2011/0044977; US 2012/0121586.

SUMMARY OF THE INVENTION

In a first aspect, the invention concerns a method for extendingprogression free survival in a HER2-positive breast cancer patientpopulation by 6 months or more comprising administering Pertuzumab,Trastuzumab and chemotherapy (e.g. taxane, such as Docetaxel) to thepatients in the population. Optionally the method results in anobjective response rate of 80% or more in the patients in thepopulation. The breast cancer is optionally metastatic or locallyrecurrent, unresectable breast cancer, or de novo Stage IV disease. Inone embodiment, the patients in the population: have not receivedprevious treatment or have relapsed after adjuvant therapy, have a leftventricular ejection fraction (LVEF) of ≥50% at baseline, and/or have anEastern Cooperative Oncology Group performance status (ECOG PS) of 0or 1. Optionally, the HER2-positive breast cancer is defined asimmunohistochemistry (IHC) 3+ and/or fluorescence in situ hybridization(FISH) amplification ratio ≥2.0. Optionally, the method reduces the riskof death by about 34% or more relative to a patient treated withTrastuzumab and the chemotherapy.

In another aspect, the invention concerns a method of combining two HER2antibodies to treat HER2-positive cancer without increasing cardiactoxicity in a HER2-positive cancer patient population, comprisingadministering Pertuzumab, Trastuzumab, and chemotherapy to the patientsin the population. Optionally, cardiac toxicity in the patientpopulation is monitored for incidence of symptomatic left ventricularsystolic dysfunction (LVSD) or congestive heart failure (CHF), or fordecrease in left ventricular ejection fraction (LVEF). The HER2-positivecancer is optionally, breast cancer, for example metastatic or locallyrecurrent, unresectable breast cancer, or de novo Stage IV disease.

In another aspect, the invention concerns an article of manufacturecomprising a vial with Pertuzumab therein and a package insert, whereinthe package insert provides the safety data in Table 3 or Table 4 and/orthe efficacy data in Table 2, Table 5, FIG. 8, or FIG. 10.

The invention additionally concerns a method for making an article ofmanufacture comprising packaging together a vial with Pertuzumab thereinand a package insert, wherein the package insert provides the safetydata in Table 3 or Table 4 and/or the efficacy data in Table 2, Table 5,FIG. 8, or FIG. 10.

In a related aspect, the invention concerns a method of ensuring safeand effective use of Pertuzumab comprising packaging together a vialwith Pertuzumab therein and a package insert, wherein the package insertprovides the safety data in Table 3 or Table 4 and/or the efficacy datain Table 2, Table 5, FIG. 8, or FIG. 10.

Optionally, the article of manufacture comprises a single-dose vialcontaining about 420 mg of Pertuzumab.

Optionally, the package insert further comprises the warning box inExample 4.

Optionally, the package insert further provides the Overall Survival(OS) efficacy data in Example 9 or Table 14.

In another aspect, the invention concerns a method of treatingearly-stage HER2-positive breast cancer comprising administeringPertuzumab, Trastuzumab, and chemotherapy to a patient with the breastcancer, wherein the chemotherapy comprises anthracycline-basedchemotherapy (for example, 5-FU, epirubicin, and cyclophosphamide(FEC)), or carboplatin-based chemotherapy (for example, Docetaxel andCarboplatin). Optionally. Pertuzumab is administered concurrently withthe anthracycline-based chemotherapy or the carboplatin-basedchemotherapy. In one embodiment of this method. Pertuzumabadministration does not increase cardiac toxicity relative to thetreatment without Pertuzumab. Such treatment of early-stageHER2-positive breast cancer optionally comprises neoadjuvant or adjuvanttherapy.

The invention further concerns a method of treating HER2-positive cancerin a patient comprising co-administering a mixture of Pertuzumab andTrastuzumab from the same intravenous bag to the patient. Such methodoptionally further comprises administering chemotherapy to the patient.

In a related aspect, the invention provides an intravenous (IV) bagcontaining a stable mixture of Pertuzumab and Trastuzumab suitable foradministration to a cancer patient. The mixture is optionally in salinesolution; e.g. comprising about 0.9% NaCl or about 0.45%/NaCl. The IVbag is optionally a 250 mL 0.9% saline polyolefin or polyvinyl chlorideinfusion bag. In one embodiment, the IV bag which contains a mixture ofabout 420 mg or of about 840 mg of Pertuzumab and from about 200 mg toabout 1000 mg of Trastuzumab. In one embodiment, the mixture is stablefor up to 24 hours at 5° C. or 30° C. Stability of the mixture can beevaluated by one or more assays selected from: color, appearance andclarity (CAC), concentration and turbidity analysis, particulateanalysis, size exclusion chromatography (SEC), ion-exchangechromatography (IEC), capillary zone electrophoresis (CZE), imagecapillary isoelectric focusing (iCIEF), or potency assay.

The present invention provides a new treatment regimen for gastriccancer. In particular, the present invention concerns the treatment ofHER2-positivegastric cancer in human subjects with a combination ofTrastuzumab, Pertuzumab and at least one chemotherapy.

In one aspect, the invention concerns a method of treating HER2-positivegastric cancer in a human subject, comprising administering to thesubject Pertuzumab, Trastuzumab, and a chemotherapy.

In one aspect, the invention concerns a method of treating gastriccancer in a human subject comprising administering Pertuzumab to thesubject with gastric cancer, wherein Pertuzumab is administered at adose of 840 mg in all treatment cycles.

In another aspect the invention concerns a method of improving survivalin a human subject with HER2-positive gastric cancer, comprisingadministering to the subject Pertuzumab, Trastuzumab, and achemotherapy.

In yet another aspect, the invention concerns Pertuzumab for use in thetreatment of HER2-positive gastric cancer in a human subject incombination with Trastuzumab and a chemotherapy.

In a further aspect, the invention concerns the use of Pertuzumab in thepreparation of a medicament for the treatment of HER2-positive gastriccancer, wherein the treatment comprises administration of Pertuzumab incombination with Trastuzumab and a chemotherapy.

In a still further aspect, the invention concerns the use of Trastuzumabin the preparation of a medicament for the treatment of HER2-positivegastric cancer, wherein the treatment comprises administration ofTrastuzumab in combination with Pertuzumab and a chemotherapy.

In another aspect, the invention concerns a kit comprising a containercomprising Pertuzumab and instructions for administration of thePertuzumab to treat HER2-positive gastric cancer in a subject incombination with Trastuzumab and a chemotherapy.

In yet another aspect, the invention concerns a kit comprising acontainer comprising Trastuzumab and instructions for administration ofthe Trastuzumab to treat HER2-positivegastric cancer in a subject incombination with Pertuzumab and a chemotherapy.

In all aspects, the gastric cancer can, for example, be non-resectablelocally advanced gastric cancer, or metastatic gastric cancer, oradvanced, post-operatively recurrent gastric cancer, which may not beamenable to curative therapy by known methods. In all aspects, thegastric cancer includes adenocarcinoma of the stomach orgastroesophageal junction. In all aspects, in a particular embodimentthe patient did not receive prior anti-cancer treatment for metastaticgastric cancer. In all aspects, in a particular embodiment, thechemotherapy comprises administration of a platin and/orfluoropyrimidine. In certain embodiments, the platin is cisplatin. Inother embodiments, the fluoropyrimidine comprises capecitabine and/or5-fluorouracil (5-FU). In all aspects, the patient's HER2-positivestatus may, for example, be IHC 3+ or IHC 2+/ISH+. In all aspects, inparticular embodiments, the treatment improves survival, includingoverall survival (OS) and/or progression free survival (PFS) and/orresponse rate (RR). In all aspects, in particular embodiments, thepatient has an ECOG PS of 0-1. In all aspects, treatment cycles aregenerally separated from each other by four weeks or less, or by threeweeks or less, or by two weeks or less, or by one week or less.

In a particular aspect, the invention concerns a method of treatingHER2-positive non-resectable or metastatic adenocarcinoma of the stomachor gastroesophageal junction in a human patient who did not receiveprior chemotherapy for metastatic disease, except prior adjuvant orneoadjuvant therapy completed more than six months before the currenttreatment, comprising administering Pertuzumab, Trastuzumab, cisplatin,and capecitabine and/or fluorouracil (5-FU) to the patient in an amountto improve progression free survival (PFS) and/or overall survival (OS),wherein the patient has an ECOG PS of 0-1. In a particular embodiment,the patient did not receive prior treatment with a platin.

In another aspect, the invention concerns a method of improvingprogression free survival in a patient with HER2-positive non-resectableor metastatic adenocarcinoma of the stomach or gastroesophageal junctioncomprising administering Pertuzumab to the patient in combination withTrastuzumab and chemotherapy.

In yet a further aspect, the invention concerns a method of treatingHER2-positive breast cancer in a patient comprising administeringPertuzumab, Trastuzumab and vinorelbine to the patient. Optionally thePertuzumab and Trastuzumab are co-administered to the patient from asingle intravenous bag. The breast cancer is optionally metastatic orlocally advanced. In one embodiment, the patient has not previouslyreceived systemic non-hormonal anticancer therapy in the metastaticsetting.

In another aspect, the invention concerns a method of treatingHER2-positive breast cancer in a patient comprising administeringPertuzumab. Trastuzumab, and aromatase inhibitor (e.g. anastrazole orletrozole) to the patient. Optionally, the breast cancer is hormonereceptor-positive advanced breast cancer, wherein the hormone receptoris estrogen receptor (ER) and/or progesterone receptor (PgR), forexample. According to this embodiment of the invention, patient has notpreviously received systemic nonhormonal anticancer therapy in themetastatic setting. Moreover, the patient herein optionally receivesinduction chemotherapy (e.g. comprising taxane).

In an additional embodiment, the invention concerns a method of treatinga cancer patient comprising administering to the patient an initial doseof 840 mg of Pertuzumab followed every 3 weeks thereafter by a dose of420 mg of Pertuzumab, and further comprising re-administering an 840 mgdose of Pertuzumab to the patient if the time between two sequential 420mg doses is 6 weeks or more. Optionally, the method further comprisesadministering 420 mg of Pertuzumab every 3 weeks after there-administered 840 mg dose. In one embodiment, the cancer patient hasHER2-positive breast cancer.

In a further aspect, the invention concerns a method for treatingHER2-positive metastatic or locally recurrent breast cancer in a patientcomprising administering Pertuzumab, Trastuzumab and taxoid (e.g.Docetaxel, Paclitaxel, or nab-paclitaxel) to the patient, wherein thepatient has been previously treated with a Trastuzumab and/or lapatinibas adjuvant or neoadjuvant therapy.

In yet a further aspect, the invention concerns a method for treatinglow HER3 ovarian, primary peritoneal, or fallopian tube cancer in apatient comprising administering Pertuzumab and chemotherapy thepatient, wherein the chemotherapy comprises taxoid (e.g. paclitaxel) ortopotecan.

In an additional aspect, the invention concerns a method for treatinglow HER3 ovarian, primary peritoneal, or fallopian tube cancer in apatient comprising administering Pertuzumab and chemotherapy to thepatient, wherein the low HER3 cancer expresses HER3 mRNA at aconcentration ratio equal or lower than about 2.81 as assessed bypolymerase chain reaction (PCR). In one embodiment, the chemotherapycomprises gemcitabine, carboplatin, paclitaxel, docetaxel, topotecan, orpegylated liposomal doxorubicin (PLD). Optionally, the chemotherapycomprises paclitaxel or topotecan. In one embodiment, the cancer isepithelial ovarian cancer that is platinum-resistant orplatinum-refractory.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of the HER2 protein structure, and aminoacid sequences for Domains I-IV (SEQ ID Nos. 1-4, respectively) of theextracellular domain thereof.

FIGS. 2A and 2B depict alignments of the amino acid sequences of thevariable light (V_(L)) (FIG. 2A) and variable heavy (V_(H)) (FIG. 2B)domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6,respectively); V_(L) and V_(H) domains of variant 574/Pertuzumab (SEQ IDNos. 7 and 8, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup 1; humIII, heavy subgroup III)(SEQ ID Nos. 9 and 10, respectively). Asterisks identify differencesbetween variable domains of Pertuzumab and murine monoclonal antibody2C4 or between variable domains of Pertuzumab and the human framework.Complementarity Determining Regions (CDRs) are in brackets.

FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chain(FIG. 3A; SEQ ID NO. 11) and heavy chain (FIG. 3B; SEQ ID No. 12). CDRsare shown in bold. Calculated molecular mass of the light chain andheavy chain are 23,526.22 Da and 49,216.56 Da (cysteines in reducedform). The carbohydrate moiety is attached to Asn 299 of the heavychain.

FIGS. 4A and 4B show the amino acid sequences of Trastuzumab light chain(FIG. 4A; SEQ ID NO. 13) and heavy chain (FIG. 4B; SEQ ID NO. 14),respectively. Boundaries of the variable light and variable heavydomains are indicated by arrows.

FIGS. 5A and 5B depict a variant Pertuzumab light chain sequence (FIG.5A; SEQ ID NO. 15) and a variant Pertuzumab heavy chain sequence (FIG.5B; SEQ ID NO. 16), respectively.

FIG. 6 shows the study schema in Example 1. ECOG=Eastern CooperativeOncology Group; PD=progressive disease. Notes: Trastuzumab, Pertuzumab,and Cisplatin are administered by IV infusion on Day 1 of each 3-weekcycle. Capecitabine is administered orally twice daily, from the eveningof Day 1 to the morning of Day 15 of each 3-week cycle. (a)HER2-positive tumor defined as either IHC 3+ or IHC 2+ in combinationwith ISH+(i.e., IHC 3+/ISH+ or ICH 2+/ISH+); (b) Trastuzumab at aloading dose of 8 mg/kg for Cycle 1 and a dose of 6 mg/kg for subsequentcycles; (c) Pertuzumab on Day 1 of each cycle, at a loading dose of 840mg for Cycle 1 and a dose of 420 mg for Cycles 2-6.

FIG. 7 depicts enrollment, intent-to-treat and safety populations, andpatient withdrawals in the study in Example 3.

FIG. 8 is a Kaplan-Meier Curve of Progression-Free Survival (PFS) asassessed by an Independent Review Facility (IRF) for the study inExample 3.

FIG. 9 depicts PFS by Patient Subgroup for the study in Example 3.

FIG. 10 depicts overall survival for the study in Example 3.

FIG. 11 is an overview of the dosing schedule in HER2-positive,neoadjuvant breast cancer, patients with low cardiac risk factors inExample 5. Additional radiotherapy, hormonal therapy and chemotherapypost surgery and during adjuvant Trastuzumab treatment were allowed ifconsidered necessary by the investigator.

FIG. 12 depicts mean change in LVEF (central readings) for the study inExample 5.

FIG. 13 shows pathological complete response (pCR) for the study inExample 5.

FIG. 14 depicts pathological complete response by hormone receptorstatus in the Example 5 study.

FIG. 15 depicts Pertuzumab SEC profile of Pertuzumab/Trastuzumab mixture(840 mg) at 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2)Time=24 hrs. Expanded view; full view (inset).

FIG. 16 shows Trastuzumab SEC profile of Pertuzumab/Trastuzumab mixture(840 mg) at 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2)Time=24 hrs. Expanded view; full view (inset).

FIG. 17 shows Pertuzumab IEC profile of Pertuzumab/Trastuzumab mixtureat 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2) Time=24hrs. Full view.

FIG. 18 depicts Trastuzumab IEC profile of Pertuzumab/Trastuzumabmixture at 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2)Time=24 hrs. Expanded view; Full view (inset).

FIG. 19 depicts CE-SDS LIF non-reduced profile of Pertuzumab/Trastuzumabmixture at 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2)Time=24 hrs. Expanded view.

FIG. 20 shows CE-SDS LIF reduced profile of Pertuzumab/Trastuzumabmixture at 30° C. in 0.9% saline PO IV infusion bags (1) Time=0; (2)Time=24 hrs. Expanded view.

FIG. 21 is CZE of Pertuzumab/Trastuzumab mixture at 30° C. in 0.9%saline PO IV infusion bags (1) Time=0; (2) Time=24 hrs. Full view.

FIG. 22 shows iCIEF of Pertuzumab/Trastuzumab mixture at 30° C. in 0.9%saline PO IV infusion bags (1) Time=0; (2) Time=24 hours. Full view.

FIG. 23 shows potency dose response curves (μg/mL versus RFU) ofPertuzumab/Trastuzumab mixture, Pertuzumab alone, and Trastuzumab alonein 0.9% saline PO IV infusion bags (1) Time=0; (2) Time=24 hours.

FIG. 24 depicts Pertuzumab SEC profile of Pertuzumab/Trastuzumab mixture(1560 mg) in 0.9% saline IV infusion bags (1) PO 5° C. T0; (2) PO 5° C.T24 hrs. (3) PO 30° C. T0; (4) PO 30° C. T24 hrs; (5) PVC 5° C. T0; (6)PVC 5° C. T24 hrs; (7) PVC 30° C. T0; (8) PVC 30° C. T24 hrs. Expandedview; full view (inset).

FIG. 25 shows Trastuzumab SEC profile of Pertuzumab/Trastuzumab mixture(1560 mg) in 0.9% saline IV infusion bags (1) PO 5° C. T0; (2) PO 5° C.T24 hrs; (3) PO 30° C. T0; (4) PO 30° C. T24 hrs; (5) PVC 5° C. T0; (6)PVC 5° C. T24 hrs; (7) PVC 30° C. T0; (8) PVC 30° C. T24 hrs. Expandedview; full view (inset).

FIG. 26 shows Pertuzumab IEC (Pertuzumab-fast) profile ofPertuzumab/Trastuzumab mixture (1560 mg) in 0.9% saline IV infusion bags(1) PO 5° C. T0; (2) PO 5° C. T24 hrs; (3) PO 30° C. T0; (4) PO 30° C.T24 hrs; (5) PVC 5° C. T0; (6) PVC 5° C. T24 hrs; (7) PVC 30° C. T0; (8)PVC 300C T24 hrs. Full view.

FIG. 27 shows Trastuzumab IEC profile of Pertuzumab/Trastuzumab mixture(1560 mg) in 0.9% saline IV infusion bags (1) PO 50C T0; (2) PO 50° C.T24 hrs; (3) PO 30° C. T0; (4) PO 30° C. T24 hrs; (5) PVC 5° C. T0; (6)PVC 5° C. T24 hrs; (7) PVC 30° C. T0; (8) PVC 30° C. T24 hrs. Full view.

FIG. 28 depicts study schema for Example 7.

FIG. 29 shows study design for Example 8.

FIG. 30 shows study design for Part 1 of Example 11.

FIG. 31 shows study design for Part 2 of Example 11.

FIG. 32 shows the samples taken and time points for the phase IIagastric cancer (GC) study in Example 1.

FIG. 33 shows the demographics of the patient population in the two armsof the GC study, treated with 420 mg (Arm A) or 840 mg (Arm B) ofPertuzumab.

FIG. 34 shows the GC history of the patients in Arms A and B,respectively.

FIG. 35 shows the GC patient disposition in Arms A and B, respectively.

FIG. 36 shows the Overall Response Rate in Arms A and B, respectively,of the GC study.

FIG. 37 shows the results of Pertuzumab Day 42 concentration assessmentin gastric cancer (GC) versus metastatic breast cancer (MBC). Day 42Ctrough is ˜37% lower in GC (JOSHUA 840/420 mg) vs. MBC (CLEO 840/420mg). JOSHUA 840/420 mg and 840/840 mg regimens both result in Day 42Ctrough ≥20 μg/mL in 90)/o of patients. JOSHUA 840/840 mg regimenresults in Day 42 Ctrough in GC comparable to that observed in MBC (CLEO840/420 mg)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Glossary of some abbreviations used herein: adverse drug reaction (ADR),adverse event (AE), alkaline phosphatase (ALP), absolute neutrophilcount (ANC), area under the concentration-time curve (AUC), capillaryzone electrophoresis (CZE), color, appearance and clarity (CAC),CLinical Evaluation Of Pertuzumab And TRAstuzumab (CLEOPATRA),confidence interval (CI), chromogenic in situ hybridization (CISH),maximum concentration (Cm), complete response (CR), case report form(CRF), computed tomography (CT), common terminology criteria for adverseevents (CTCAE), Docetaxel (D), dose limiting toxicity (DLT), ethicscommittee (EC), epirubicin, cisplatin, and 5-fluorouracil (ECF),echocardiogram (ECHO), epidermal growth factor receptor (EGFR), EuropeanUnion (EU), estrogen receptor (ER), 5-fluorouracil, methotrexate, anddoxorubicin (FAMTX), fluorescence in situ hybridization (FISH),5-fluorouracil (5-FU), hazard ratio (HR), human epidermal growth factorreceptor (EGFR), gastric cancer (GC), good clinical practice (GCP),human epidermal growth factor receptor 2 (HER2), ion exchangechromatography (IEC), immunohistochemistry (IHC), independent reviewfacility (IRF), institutional review board (IRB), in situ hybridization(ISH), intravenous (IV), image capillary isoelectric focusing (iCIEF),left ventricular ejection fraction (LVEF), mitomycin C, cisplatin, and5-fluorouracil (MCF), magnetic resonance image (MRI), metastatic breastcancer (MBC), multiple-gated acquisition (MUGA), not significant (NS),overall survival (OS), pathological complete response (pCR), polyolefin(PO), polyvinyl chloride (PVC), progressive disease (PD), progressionfree survival (PFS), pharmacokinetic (PK), partial response (PR),progesterone receptor (PgR), response evaluation criteria in solidtumors (RECIST), serious adverse event (SAE), size exclusionchromatography (SEC), stable disease (SD), study management team (SMT),sterile water for injection (SWFI), time to maximum plasma concentration(t_(max)), upper limit of normal (ULN).

I. Definitions

The term “chemotherapy” as used herein refers to treatment comprisingthe administration of a chemotherapy, as defined hereinbelow.

“Survival” refers to the patient remaining alive, and includes overallsurvival as well as progression free survival.

“Overall survival” or “OS” refers to the patient remaining alive for adefined period of time, such as 1 year, 5 years, etc from the time ofdiagnosis or treatment. For the purposes of the clinical trial describedin the example, overall survival (OS) is defined as the time from thedate of randomization of patient population to the date of death fromany cause.

“Progression free survival” or “PFS” refers to the patient remainingalive, without the cancer progressing or getting worse. For the purposeof the clinical trial described in the example, progression freesurvival (PFS) is defined as the time from randomization of studypopulation to the first documented progressive disease, or unmanageabletoxicity, or death from any cause, whichever occurs first. Diseaseprogression can be documented by any clinically accepted methods, suchas, for example, radiographical progressive disease, as determined byResponse Evaluation Criteria in Solid Tumors (RECIST) (Therasse et al.,J Natl Ca Inst 2000; 92(3):205-216), carcinomatous meningitis diagnosedby cytologic evaluation of cerebral spinal fluid, and/or medicalphotography to monitor chest wall recurrences of subcutaneous lesions.

By “extending survival” is meant increasing overall or progression freesurvival in a patient treated in accordance with the present inventionrelative to an untreated patient and/or relative to a patient treatedwith one or more approved anti-tumor agents, but not receiving treatmentin accordance with the present invention. In a particular example,“extending survival” means extending progression-free survival (PFS)and/or overall survival (OS) of cancer patients receiving thecombination therapy of the present invention (e.g. treatment with acombination of Pertuzumab, Trastuzumab and a chemotherapy) relative topatients treated with Trastuzumab and the chemotherapy only. In anotherparticular example, “extending survival” means extendingprogression-free survival (PFS) and/or overall survival (OS) of cancerpatients receiving the combination therapy of the present invention(e.g. treatment with a combination of Pertuzumab, Trastuzumab and achemotherapy) relative to patients treated with Pertuzumab and thechemotherapy only.

An “objective response” refers to a measurable response, includingcomplete response (CR) or partial response (PR).

By “complete response” or “CR” is intended the disappearance of allsigns of cancer in response to treatment. This does not always mean thecancer has been cured.

“Partial response” or “PR” refers to a decrease in the size of one ormore tumors or lesions, or in the extent of cancer in the body, inresponse to treatment.

A “HER receptor” is a receptor protein tyrosine kinase which belongs tothe HER receptor family and includes EGFR. HER2. HER3 and HER4receptors. The HER receptor will generally comprise an extracellulardomain, which may bind an HER ligand and/or dimerize with another HERreceptor molecule; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.The HER receptor may be a “native sequence” HER receptor or an “aminoacid sequence variant” thereof. Preferably the HER receptor is nativesequence human HER receptor.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234(1986) (Genebank accession number X03363). The term “erbB2” refers tothe gene encoding human ErbB2 and “neu” refers to the gene encoding ratp185^(neu). Preferred HER2 is native sequence human HER2.

Herein, “HER2 extracellular domain” or “HER2 ECD” refers to a domain ofHER2 that is outside of a cell, either anchored to a cell membrane, orin circulation, including fragments thereof. The amino acid sequence ofHER2 is shown in FIG. 1. In one embodiment, the extracellular domain ofHER2 may comprise four domains: “Domain I” (amino acid residues fromabout 1-195; SEQ ID NO:1). “Domain II” (amino acid residues from about196-319; SEQ ID NO:2), “Domain 111” (amino acid residues from about320-488: SEQ ID NO:3), and “Domain IV” (amino acid residues from about489-630; SEQ ID NO:4) (residue numbering without signal peptide). SeeGarrett et al. Mol. Cell. 11: 495-505 (2003). Cho et al. Nature 421:756-760 (2003). Franklin et al. Cancer Cell 5:317-328 (2004), andPlowman et al. Proc. Natl. Acad. Sci. 90:1746-1750 (1993), as well asFIG. 6 herein.

“HER3” or “ErbB3” herein refer to the receptor as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989).

A “low HER3” cancer is one which expresses HER3 at a level less then themedian level for HER3 expression in the cancer type. In one embodiment,the low HER3 cancer is epithelial ovarian, peritoneal, or fallopian tubecancer. HER3 DNA, protein, and/or mRNA level in the cancer can beevaluated to determine whether the cancer is a low HER3 cancer. See, forexample, U.S. Pat. No. 7,981,418 for additional information about lowHER3 cancer. Optionally, a HER3 mRNA expression assay is performed inorder to determine that the cancer is a low HER3 cancer. In oneembodiment, HER3 mRNA level in the cancer is evaluated, e.g. usingpolymerase chain reaction (PCR), such as quantitative reversetranscription PCR (qRT-PCR). Optionally, the cancer expresses HER3 at aconcentration ratio equal or lower than about 2.81 as assessed qRT-PCR.e.g. using a COBAS z480® instrument.

A “HER dimer” herein is a noncovalently associated dimer comprising atleast two HER receptors. Such complexes may form when a cell expressingtwo or more HER receptors is exposed to an HER ligand and can beisolated by immunoprecipitation and analyzed by SDS-PAGE as described inSliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), forexample. Other proteins, such as a cytokine receptor subunit (e.g.gp130) may be associated with the dimer. Preferably, the HER dimercomprises HER2.

A “HER heterodimer” herein is a noncovalently associated heterodimercomprising at least two different HER receptors, such as EGFR-HER2.HER2-HER3 or HER2-HER4 heterodimers.

A “HER antibody” is an antibody that binds to a HER receptor.Optionally, the HER antibody further interferes with HER activation orfunction. Preferably, the HER antibody binds to the HER2 receptor. HER2antibodies of interest herein are Pertuzumab and Trastuzumab.

“HER activation” refers to activation, or phosphorylation, of any one ormore HER receptors. Generally, HER activation results in signaltransduction (e.g. that caused by an intracellular kinase domain of aHER receptor phosphorylating tyrosine residues in the HER receptor or asubstrate polypeptide). HER activation may be mediated by HER ligandbinding to a HER dimer comprising the HER receptor of interest. HERligand binding to a HER dimer may activate a kinase domain of one ormore of the HER receptors in the dimer and thereby results inphosphorylation of tyrosine residues in one or more of the HER receptorsand/or phosphorylation of tyrosine residues in additional substratepolypeptides(s), such as Akt or MAPK intracellular kinases.

“Phosphorylation” refers to the addition of one or more phosphategroup(s) to a protein, such as a HER receptor, or substrate thereof.

An antibody which “inhibits HER dimerization” is an antibody whichinhibits, or interferes with, formation of a HER dimer. Preferably, suchan antibody binds to HER2 at the heterodimeric binding site thereof. Themost preferred dimerization inhibiting antibody herein is Pertuzumab orMAb 2C4. Other examples of antibodies which inhibit HER dimerizationinclude antibodies which bind to EGFR and inhibit dimerization thereofwith one or more other HER receptors (for example EGFR monoclonalantibody 806, MAb 806, which binds to activated or “untethered” EGFR;see Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)); antibodieswhich bind to HER3 and inhibit dimerization thereof with one or moreother HER receptors; and antibodies which bind to HER4 and inhibitdimerization thereof with one or more other HER receptors.

A “HER2 dimerization inhibitor” is an agent that inhibits formation of adimer or heterodimer comprising HER2.

A “heterodimeric binding site” on HER2, refers to a region in theextracellular domain of HER2 that contacts, or interfaces with, a regionin the extracellular domain of EGFR, HER3 or HER4 upon formation of adimer therewith. The region is found in Domain II of HER2 (SEQ ID NO:15). Franklin et al. Cancer Cell 5:317-328 (2004).

A HER2 antibody that “binds to a heterodimeric binding site” of HER2,binds to residues in Domain II (SEQ ID NO: 2) and optionally also bindsto residues in other of the domains of the HER2 extracellular domain,such as domains I and III, SEQ ID NOs: 1 and 3), and can stericallyhinder, at least to some extent, formation of a HER2-EGFR, HER2-HER3, orHER2-HER4 heterodimer. Franklin et al. Cancer Cell 5:317-328 (2004)characterize the HER2-Pertuzumab crystal structure, deposited with theRCSB Protein Data Bank (ID Code IS78), illustrating an exemplaryantibody that binds to the heterodimeric binding site of HER2.

An antibody that “binds to domain II” of HER2 binds to residues indomain II (SEQ ID NO: 2) and optionally residues in other domain(s) ofHER2, such as domains I and III (SEQ ID NOs: 1 and 3, respectively).Preferably the antibody that binds to domain II binds to the junctionbetween domains I, II and III of HER2.

For the purposes herein, “Pertuzumab” and “rhuMAb 2C4”, which are usedinterchangeably, refer to an antibody comprising the variable light andvariable heavy amino acid sequences in SEQ ID NOs: 7 and 8,respectively. Where Pertuzumab is an intact antibody, it preferablycomprises an IgG1 antibody; in one embodiment comprising the light chainamino acid sequence in SEQ ID NO: 11 or 15, and heavy chain amino acidsequence in SEQ ID NO: 12 or 16. The antibody is optionally produced byrecombinant Chinese Hamster Ovary (CHO) cells. The terms “Pertuzumab”and “rhuMAb 2C4” herein cover biosimilar versions of the drug with theUnited States Adopted Name (USAN) or International Nonproprietary Name(INN): Pertuzumab.

For the purposes herein, “Trastuzumab” and “rhuMAb4D5”, which are usedinterchangeably, refer to an antibody comprising the variable light andvariable heavy amino acid sequences from within SEQ ID Nos: 13 and 14,respectively. Where Trastuzumab is an intact antibody, it preferablycomprises an IgG1 antibody; in one embodiment comprising the light chainamino acid sequence of SEQ ID NO: 13 and the heavy chain amino acidsequence of SEQ ID NO: 14. The antibody is optionally produced byChinese Hamster Ovary (CHO) cells. The terms “Trastuzumab” and“rhuMAb4D5” herein cover biosimilar versions of the drug with the UnitedStates Adopted Name (USAN) or International Nonproprietary Name (INN):Trastuzumab.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies), and antibodyfragments, so long as they exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). Humanized HER2antibodies specifically include Trastuzumab (HERCEPTIN®) as described inTable 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein byreference and as defined herein; and humanized 2C4 antibodies such asPertuzumab as described and defined herein.

An “intact antibody” herein is one which comprises two antigen bindingregions, and an Fc region. Preferably, the intact antibody has afunctional Fc region.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragment(s).

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed.Public Health Service. National Institutes of Health. Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1). 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

Unless indicated otherwise, herein the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided intoAsubclasses@(isotypes), e.g., IgG1. IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofantibodies are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

A “naked antibody” is an antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety or radiolabel.

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al. J.Mol. Biol. 226:889-896 (1)992).

A “deaminated” antibody is one in which one or more asparagine residuesthereof has been derivatized, e.g. to an aspartic acid, a succinimide,or an iso-aspartic acid.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth.

“Gastric cancer” specifically includes metastatic or locally advancednon-resectable gastric cancer, including, without limitation,histologically confirmed adenocarcinoma of the stomach orgastroesophageal junction with inoperable (non-resectable) locallyadvanced or metastatic disease, not amenable to curative therapy, andpost-operatively recurrent advanced gastric cancer, such asadenocarcinoma of the stomach or gastroesophageal junction, when theintent of the surgery was to cure the disease.

An “advanced” cancer is one which has spread outside the site or organof origin, either by local invasion or metastasis. Accordingly, the term“advanced” cancer includes both locally advanced and metastatic disease.

A “refractory” cancer is one which progresses even though an anti-tumoragent, such as a chemotherapy, is being administered to the cancerpatient. An example of a refractory cancer is one which is platinumrefractory.

A “recurrent” cancer is one which has regrown, either at the initialsite or at a distant site, after a response to initial therapy, such assurgery.

A “locally recurrent” cancer is cancer that returns after treatment inthe same place as a previously treated cancer.

A “non-resectable” or “unresectable” cancer is not able to be removed(resected) by surgery.

“Early-stage breast cancer” herein refers to breast cancer that has notspread beyond the breast or the axillary lymph nodes. Such cancer isgenerally treated with neoadjuvant or adjuvant therapy.

“Neoadjuvant therapy” refers to systemic therapy given prior to surgery.

“Adjuvant therapy” refers to systemic therapy given after surgery.

“Metastatic” cancer refers to cancer which has spread from one part ofthe body (e.g. the breast) to another part of the body.

Herein, a “patient” or “subject” is a human patient. The patient may bea “cancer patient.” i.e. one who is suffering or at risk for sufferingfrom one or more symptoms of cancer, in particular gastric or breastcancer.

A “patient population” refers to a group of cancer patients. Suchpopulations can be used to demonstrate statistically significantefficacy and/or safety of a drug, such as Pertuzumab.

A “relapsed” patient is one who has signs or symptoms of cancer afterremission. Optionally, the patient has relapsed after adjuvant orneoadjuvant therapy.

A cancer or biological sample which “displays HER expression,amplification, or activation” is one which, in a diagnostic test,expresses (including overexpresses) a HER receptor, has amplified HERgene, and/or otherwise demonstrates activation or phosphorylation of aHER receptor.

A cancer or biological sample which “displays HER activation” is onewhich, in a diagnostic test, demonstrates activation or phosphorylationof a HER receptor. Such activation can be determined directly (e.g. bymeasuring HER phosphorylation by ELISA) or indirectly (e.g. by geneexpression profiling or by detecting HER heterodimers, as describedherein).

A cancer cell with “HER receptor overexpression or amplification” is onewhich has significantly higher levels of a HER receptor protein or genecompared to a noncancerous cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. HER receptor overexpression oramplification may be determined in a diagnostic or prognostic assay byevaluating increased levels of the HER protein present on the surface ofa cell (e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of HER-encoding nucleic acid in thecell, e.g. via in situ hybridization (ISH), including fluorescent insitu hybridization (FISH; see WO98/45479 published October. 1998) andchromogenic in situ hybridization (CISH; see, e.g. Tanner et al., Am. J.Pathol. 157(5): 1467-1472 (2000); Bella et al., J. Clin. Oncol. 26: (May20 suppl; abstr 22147) (2008)), southern blotting, or polymerase chainreaction (PCR) techniques, such as quantitative real time PCR (qRT-PCR).One may also study HER receptor overexpression or amplification bymeasuring shed antigen (e.g., HER extracellular domain) in a biologicalfluid such as serum (see, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12,1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issuedMar. 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)).Aside from the above assays, various in vivo assays are available to theskilled practitioner. For example, one may expose cells within the bodyof the patient to an antibody which is optionally labeled with adetectable label, e.g. a radioactive isotope, and binding of theantibody to cells in the patient can be evaluated, e.g. by externalscanning for radioactivity or by analyzing a biopsy taken from a patientpreviously exposed to the antibody.

A “HER2-positive” cancer comprises cancer cells which have higher thannormal levels of HER2. Examples of HER2-positive cancer includeHER2-positive breast cancer and HER2-positive gastric cancer.Optionally, HER2-positive cancer has an immunohistochemistry (IHC) scoreof 2+ or 3+ and/or an in situ hybridization (ISH) amplification ratio≥2.0.

Herein, an “anti-tumor agent” refers to a drug used to treat cancer.Non-limiting examples of anti-tumor agents herein include chemotherapyagents. HER dimerization inhibitors, HER antibodies, antibodies directedagainst tumor associated antigens, anti-hormonal compounds, cytokines,EGFR-targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors,growth inhibitory agents and antibodies, cytotoxic agents, antibodiesthat induce apoptosis, COX inhibitors, farnesyl transferase inhibitors,antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf orras inhibitors, liposomal doxorubicin, topotecan, taxene, dual tyrosinekinase inhibitors, TLK286, EMD-7200, Pertuzumab, Trastuzumab, erlotinib,and bevacizumab.

The “epitope 2C4” is the region in the extracellular domain of HER2 towhich the antibody 2C4 binds. In order to screen for antibodies whichbind essentially to the 2C4 epitope, a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual. Cold Spring HarborLaboratory. Ed Harlow and David Lane (1988), can be performed.Preferably the antibody blocks 2C4's binding to HER2 by about 50% ormore. Alternatively, epitope mapping can be performed to assess whetherthe antibody binds essentially to the 2C4 epitope of HER2. Epitope 2C4comprises residues from Domain II (SEQ ID NO: 2) in the extracellulardomain of HER2. 2C4 and Pertuzumab binds to the extracellular domain ofHER2 at the junction of domains I, II and III (SEQ ID NOs: 1, 2, and 3,respectively). Franklin et al. Cancer Cell 5:317-328 (2004).

The “epitope 4D5” is the region in the extracellular domain of HER2 towhich the antibody 4D5 (ATCC CRL 10463) and Trastuzumab bind. Thisepitope is close to the transmembrane domain of HER2, and within DomainIV of HER2 (SEQ ID NO: 4). To screen for antibodies which bindessentially to the 4D5 epitope, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds essentially to the 4D5 epitope of HER2 (e.g. any one ormore residues in the region from about residue 529 to about residue 625,inclusive of the HER2 ECD, residue numbering including signal peptide).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith cancer as well as those in which cancer is to be prevented. Hence,the patient to be treated herein may have been diagnosed as havingcancer or may be predisposed or susceptible to cancer.

The term “effective amount” refers to an amount of a drug effective totreat cancer in the patient. The effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. The effectiveamount may extend progression free survival (e.g. as measured byResponse Evaluation Criteria for Solid Tumors, RECIST, or CA-125changes), result in an objective response (including a partial response,PR, or complete response. CR), increase overall survival time, and/orimprove one or more symptoms of cancer (e.g. as assessed by FOSI).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰. Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², p³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapy” is use of a chemical compound useful in the treatmentof cancer. Examples of chemotherapeutic agents, used in chemotherapy,include alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; TLK 286(TELCYTA™); acetogenins (especially bullatacin and bullatacinone);delta-9-tetrahydrocannabinol (dronabinol. MARINOL®); beta-lapachone;lapachol; colchicines; betulinic acid; a camptothecin (including thesynthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan,CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin);bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesinand bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid;teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimustine; bisphosphonates, such as clodronate; antibiotics suchas the enediyne antibiotics (e.g., calicheamicin, especiallycalicheamicin gammal II and calicheamicin omegaI1 (see, e.g., Agnew.Chem Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such asannamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1,epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin,including dynemicin A, an esperamicin, neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin),esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; folic acid analogues such asdenopterin, pteropterin, and trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide,mitotane, and trilostane; folic acid replenisher such as folinic acid(leucovorin); aceglatone; anti-folate anti-neoplastic agents such asALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such asmethotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and itsprodrugs such as UFT, S-1 and capecitabine, and thymidylate synthaseinhibitors and glycinamide ribonucleotide formyltranferase inhibitorssuch as raltitrexed (TOMUDEX®, TDX); inhibitors of dihydropyrimidinedehydrogenase such as eniluracil; aldophosphamide glycoside;aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidainine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;PSK7 polysaccharide complex (JHS Natural Products. Eugene, Oreg.);razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine(ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine(GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs orplatinum-based analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); vinca alkaloid; vinorelbine (NAVELBINE®);novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);retinoids such as retinoic acid; pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone,and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; aromatase inhibitors; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in abherant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras. and epidermal growth factor receptor(EGF-R); vaccines such as gene therapy vaccines, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN®rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A “taxane” is a chemotherapy which inhibits mitosis and interferes withmicrotubules. Examples of taxanes include Paclitaxel (TAXOL®;Bristol-Myers Squibb Oncology, Princeton, N.J.); cremophor-free,albumin-engineered nanoparticle formulation of paclitaxel ornab-paclitaxel (ABRAXANE™; American Pharmaceutical Partners, Schaumberg,Ill.); and Docetaxel (TAXOTERE®; Rhône-Poulenc Rorer. Antony, France).

An “anthacycline” is a type of antibiotic that comes from the fungusStreptococcus peucetius, examples include: Daunorubicin, Doxorubicin,and Epirubicin, etc.

“Anthracycline-based chemotherapy” refers to a chemotherapy regimen thatconsists of or include one or more anthracycline. Examples include 5-FU,epirubicin, and cyclophosphamide (FEC); 5-FU, doxorubicin, andcyclophosphamide (FAC); doxorubicin and cyclophosphamide (AC);epirubicin and cyclophosphamide (EC); etc.

For the purposes herein, “carboplatin-based chemotherapy” refers to achemotherapy regimen that consists of or includes one or moreCarboplatins. An example is TCH (Docetaxel/TAXOL®, Carboplatin, andTrastuzumabiHERCEPTIN®).

An “aromatase inhibitor” inhibits the enzyme aromatase, which regulatesestrogen production in the adrenal glands. Examples of aromataseinhibitors include: 4(5)-imidazoles, aminoglutethimide. MEGASE®megestrol acetate. AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole. FEMARA® letrozole, and ARIMIDEX® anastrozole. In oneembodiment, the aromatase inhibitor herein is letrozole or anastrozole.

An “antimetabolite chemotherapy” is use of an agent which isstructurally similar to a metabolite, but can not be used by the body ina productive manner. Many antimetabolite chemotherapy interferes withthe production of the nucleic acids, RNA and DNA. Examples ofantimetabolite chemotherapeutic agents include gemcitabine (GEMZAR®),5-fluorouracil (5-FU), capecitabine (XELODAT™), 6-mercaptopurine,methotrexate, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosineARA-C cytarabine (CYTOSAR-U®), dacarbazine (DTIC-DOME®), azocytosine,deoxycytosine, pyridmidene, fludarabine (FLUDARA®), cladrabine,2-deoxy-D-glucose etc.

By “chemotherapy-resistant” cancer is meant that the cancer patient hasprogressed while receiving a chemotherapy regimen (i.e. the patient is“chemotherapy refractory”), or the patient has progressed within 12months (for instance, within 6 months) after completing a chemotherapyregimen.

The term “platin” is used herein to refer to platinum basedchemotherapy, including, without limitation, cisplatin, carboplatin, andoxaliplatin.

The term “fluoropyrimidine” is used herein to refer to an antimetabolitechemotherapy, including, without limitation, capecitabine, floxuridine,and fluorouracil (5-FU).

A “fixed” or “flat” dose of a therapeutic agent herein refers to a dosethat is administered to a human patient without regard for the weight(WT) or body surface area (BSA) of the patient. The fixed or flat doseis therefore not provided as a mg/kg dose or a mg/m² dose, but rather asan absolute amount of the therapeutic agent.

A “loading” dose herein generally comprises an initial dose of atherapeutic agent administered to a patient, and is followed by one ormore maintenance dose(s) thereof. Generally, a single loading dose isadministered, but multiple loading doses are contemplated herein.Usually, the amount of loading dose(s) administered exceeds the amountof the maintenance dose(s) administered and/or the loading dose(s) areadministered more frequently than the maintenance dose(s), so as toachieve the desired steady-state concentration of the therapeutic agentearlier than can be achieved with the maintenance dose(s).

A “maintenance” dose herein refers to one or more doses of a therapeuticagent administered to the patient over a treatment period. Usually, themaintenance doses are administered at spaced treatment intervals, suchas approximately every week, approximately every 2 weeks, approximatelyevery 3 weeks, or approximately every 4 weeks, preferably every 3 weeks.

“Infusion” or “infusing” refers to the introduction of a drug-containingsolution into the body through a vein for therapeutic purposes.Generally, this is achieved via an intravenous (IV) bag.

An “intravenous bag” or “IV bag” is a bag that can hold a solution whichcan be administered via the vein of a patient. In one embodiment, thesolution is a saline solution (e.g. about 0.9% or about 0.45% NaCl).Optionally, the IV bag is formed from polyolefin or polyvinyl chloride.

By “co-administering” is meant intravenously administering two (or more)drugs during the same administration, rather than sequential infusionsof the two or more drugs. Generally, this will involve combining the two(or more) drugs into the same IV bag prior to co-administration thereof.

“Cardiac toxicity” refers to any toxic side effect resulting fromadministration of a drug or drug combination. Cardiac toxicity can beevaluated based on any one or more of: incidence of symptomatic leftventricular systolic dysfunction (LVSD) or congestive heart failure(CHF), or decrease in left ventricular ejection fraction (LVEF).

The phrase “without increasing cardiac toxicity” for a drug combinationincluding Pertuzumab refers to an incidence of cardiac toxicity that isequal or less than that observed in patients treated with drugs otherthan Pertuzumab in the drug combination (e.g. equal or less than thatresulting from administration of Trastuzumab and the chemotherapy, e.g.Docetaxel).

A “vial” is a container suitable for holding a liquid or lyophilizedpreparation. In one embodiment, the vial is a single-use vial, e.g. a20-cc single-use vial with a stopper.

A “package insert” is a leaflet that, by order of the Food and DrugAdministration (FDA) or other Regulatory Authority, must be placedinside the package of every prescription drug. The leaflet generallyincludes the trademark for the drug, its generic name, and its mechanismof action; states its indications, contraindications, warnings,precautions, adverse effects, and dosage forms; and includesinstructions for the recommended dose, time, and route ofadministration.

The expression “safety data” concerns the data obtained in a controlledclinical trial showing the prevalence and severity of adverse events toguide the user regarding the safety of the drug, including guidance onhow to monitor and prevent adverse reactions to the drug. Table 3 andTable 4 herein provide safety data for Pertuzumab. The safety datacomprises any one or more (e.g. two, three, four or more) of the mostcommon adverse events (AEs) or adverse reactions (ADRs) in Tables 3 and4. For example, the safety data comprises information about neutropenia,febrile neutropenia, diarrhea and/or cardiac toxicity as disclosedherein.

“Efficacy data” refers to the data obtained in controlled clinical trialshowing that a drug effectively treats a disease, such as cancer.Efficacy data for Pertuzumab is provided in the examples herein. As toHER2-positive metastatic or locally recurrent, unresectable breastcancer, efficacy data for Pertuzumab is found in Table 2, Table 5, FIG.8 and FIG. 10 herein. The safety data comprises any one or more (e.g.two, three, four or more) of the primary endpoint (progression freesurvival, PFS, by IRF) and/or secondary enpoints (overall survival (OS);progression free survival (PFS) by investigator; objective response rate(ORR), including complete response (CR), partial response (PR), stabledisease (SD), and progressive disease (PD), and/or duration of response)in Table 2. Table 5. FIG. 8 and FIG. 10. For example, the efficacy datacomprises information about progression free survival (PFS) and/oroverall survival (OS) as disclosed herein.

By “stable mixture” when referring to a mixture of two or more drugs,such as Pertuzumab and Trastuzumab” means that each of the drugs in themixture essentially retains its physical and chemical stability in themixture as evaluated by one or more analytical assays. Exemplaryanalytical assays for this purpose include: color, appearance andclarity (CAC), concentration and turbidity analysis, particulateanalysis, size exclusion chromatography (SEC), ion-exchangechromatography (IEC), capillary zone electrophoresis (CZE), imagecapillary isoelectric focusing (iCIEF), and potency assay. In oneembodiment, mixture has been shown to be stable for up to 24 hours at 5°C. or 30° C.

A drug that is administered “concurrently” with one or more other drugsis administered during the same treatment cycle, on the same day oftreatment as the one or more other drugs, and, optionally, at the sametime as the one or more other drugs. For instance, for cancer therapiesgiven every 3-weeks, the concurrently administered drugs are eachadministered on day-1 of a 3-week cycle.

II. Antibody and Chemotherapy Compositions

The HER2 antigen to be used for production of antibodies may be, e.g., asoluble form of the extracellular domain of a HER2 receptor or a portionthereof, containing the desired epitope. Alternatively, cells expressingHER2 at their cell surface (e.g. NIH-3T3 cells transformed tooverexpress HER2; or a carcinoma cell line such as SK-BR-3 cells, seeStancovski et al. PNAS (USA) 88:8691-8695 (1991)) can be used togenerate antibodies. Other forms of HER2 receptor useful for generatingantibodies will be apparent to those skilled in the art.

Various methods for making monoclonal antibodies herein are available inthe art. For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), by recombinant DNA methods (U.S. Pat. No. 4,816,567).

The anti-HER2 antibodies used in accordance with the present invention,Trastuzumab and Pertuzumab, are commercially available.

(i) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Rieclunann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

U.S. Pat. No. 6,949,245 describes production of exemplary humanized HER2antibodies which bind HER2 and block ligand activation of a HERreceptor.

Humanized HER2 antibodies specifically include Trastuzumab (HERCEPTIN®)as described in Table 3 of U.S. Pat. No. 5,821,337 expresslyincorporated herein by reference and as defined herein; and humanized2C4 antibodies such as Pertuzumab as described and defined herein.

The humanized antibodies herein may, for example, comprise nonhumanhypervariable region residues incorporated into a human variable heavydomain and may further comprise a framework region (FR) substitution ata position selected from the group consisting of 69H, 71H and 73Hutilizing the variable domain numbering system set forth in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991). Inone embodiment, the humanized antibody comprises FR substitutions at twoor all of positions 69H, 71H and 73H.

An exemplary humanized antibody of interest herein comprises variableheavy domain complementarity determining residues GFTFTDYTMX (SEQ ID NO:17), where X is preferably D or S; DVNPNSGGSIYNQRFKG (SEQ ID NO:18);and/or NLGPSFYFDY (SEQ ID NO:19), optionally comprising amino acidmodifications of those CDR residues, e.g. where the modificationsessentially maintain or improve affinity of the antibody. For example,an antibody variant for use in the methods of the present invention mayhave from about one to about seven or about five amino acidsubstitutions in the above variable heavy CDR sequences. Such antibodyvariants may be prepared by affinity maturation, e.g., as describedbelow.

The humanized antibody may comprise variable light domaincomplementarity determining residues KASQDVSIGVA (SEQ ID NO:20);SASYX¹X²X³, where X¹ is preferably R or L. X² is preferably Y or E, andX³ is preferably T or S (SEQ ID NO:21); and/or QQYYIYPYT (SEQ ID NO:22),e.g. in addition to those variable heavy domain CDR residues in thepreceding paragraph. Such humanized antibodies optionally comprise aminoacid modifications of the above CDR residues, e.g. where themodifications essentially maintain or improve affinity of the antibody.For example, the antibody variant of interest may have from about one toabout seven or about five amino acid substitutions in the above variablelight CDR sequences. Such antibody variants may be prepared by affinitymaturation, e.g., as described below.

The present application also contemplates affinity matured antibodieswhich bind HER2.

The parent antibody may be a human antibody or a humanized antibody,e.g., one comprising the variable light and/or variable heavy sequencesof SEQ ID Nos. 7 and 8, respectively (i.e. comprising the VL and/or VHof Pertuzumab). An affinity matured variant of Pertuzumab preferablybinds to HER2 receptor with an affinity superior to that of murine 2C4or Pertuzumab (e.g. from about two or about four fold, to about 100 foldor about 1000 fold improved affinity, e.g. as assessed using aHER2-extracellular domain (ECD) ELISA). Exemplary variable heavy CDRresidues for substitution include H28, H30, H34, H35, H64, H96, H99, orcombinations of two or more (e.g. two, three, four, five, six, or sevenof these residues). Examples of variable light CDR residues foralteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 orcombinations of two or more (e.g. two to three, four, five or up toabout ten of these residues).

Humanization of murine 4D5 antibody to generate humanized variantsthereof, including Trastuzumab, is described in U.S. Pat. Nos.5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and 6,800,738, aswell as Carter et al. PNAS (USA), 89:4285-4289 (1992). HuMAb4D5-8(Trastuzumab) bound HER2 antigen 3-fold more tightly than the mouse 4D5antibody, and had secondary immune function (ADCC) which allowed fordirected cytotoxic activity of the humanized antibody in the presence ofhuman effector cells. HuMAb4D5-8 comprised variable light (V_(L)) CDRresidues incorporated in a V_(L) κ subgroup I consensus framework, andvariable heavy (V_(H)) CDR residues incorporated into a V_(H) subgroupIII consensus framework. The antibody further comprised framework region(FR) substitutions as positions: 71, 73, 78, and 93 of the V_(H) (Kabatnumbering of FR residues; and a FR substitution at position 66 of theV_(L) (Kabat numbering of FR residues). Trastuzumab comprises non-Aallotype human γ 1 Fc region.

Various forms of the humanized antibody or affinity matured antibody arecontemplated. For example, the humanized antibody or affinity maturedantibody may be an antibody fragment. Alternatively, the humanizedantibody or affinity matured antibody may be an intact antibody, such asan intact IgG1 antibody.

(ii) Pertuzumab Compositions

In one embodiment of a HER2 antibody composition, the compositioncomprises a mixture of a main species Pertuzumab antibody and one ormore variants thereof. The preferred embodiment herein of a Pertuzumabmain species antibody is one comprising the variable light and variableheavy amino acid sequences in SEQ ID Nos. 7 and 8, and most preferablycomprising a light chain amino acid sequence of SEQ ID No. 11, and aheavy chain amino acid sequence of SEQ ID No. 12 (including deamidatedand/or oxidized variants of those sequences). In one embodiment, thecomposition comprises a mixture of the main species Pertuzumab antibodyand an amino acid sequence variant thereof comprising an amino-terminalleader extension. Preferably, the amino-terminal leader extension is ona light chain of the antibody variant (e.g. on one or two light chainsof the antibody variant). The main species HER2 antibody or the antibodyvariant may be an full length antibody or antibody fragment (e.g. Fab ofF(ab=)2 fragments), but preferably both are full length antibodies. Theantibody variant herein may comprise an amino-terminal leader extensionon any one or more of the heavy or light chains thereof. Preferably, theamino-terminal leader extension is on one or two light chains of theantibody. The amino-terminal leader extension preferably comprises orconsists of VHS-. Presence of the amino-terminal leader extension in thecomposition can be detected by various analytical techniques including,but not limited to, N-terminal sequence analysis, assay for chargeheterogeneity (for instance, cation exchange chromatography or capillaryzone electrophoresis), mass spectrometry, etc. The amount of theantibody variant in the composition generally ranges from an amount thatconstitutes the detection limit of any assay (preferably N-terminalsequence analysis) used to detect the variant to an amount less than theamount of the main species antibody. Generally, about 20% or less (e.g.from about 1% to about 15%, for instance from 5% to about 15%) of theantibody molecules in the composition comprise an amino-terminal leaderextension. Such percentage amounts are preferably determined usingquantitative N-terminal sequence analysis or cation exchange analysis(preferably using a high-resolution, weak cation-exchange column, suchas a PROPAC WCX-10™ cation exchange column). Aside from theamino-terminal leader extension variant, further amino acid sequencealterations of the main species antibody and/or variant arecontemplated, including but not limited to an antibody comprising aC-terminal lysine residue on one or both heavy chains thereof, adeamidated antibody variant, etc.

Moreover, the main species antibody or variant may further compriseglycosylation variations, non-limiting examples of which includeantibody comprising a G1 or G2 oligosaccharide structure attached to theFc region thereof, antibody comprising a carbohydrate moiety attached toa light chain thereof (e.g. one or two carbohydrate moieties, such asglucose or galactose, attached to one or two light chains of theantibody, for instance attached to one or more lysine residues),antibody comprising one or two non-glycosylated heavy chains, orantibody comprising a sialidated oligosaccharide attached to one or twoheavy chains thereof etc.

The composition may be recovered from a genetically engineered cellline, e.g. a Chinese Hamster Ovary (CHO) cell line expressing the HER2antibody, or may be prepared by peptide synthesis.

For more information regarding exemplary Pertuzumab compositions, seeU.S. Pat. Nos. 7,560,111 and 7,879,325 as well as US 2009/0202546A1.

(iii) Trastuzumab Compositions

The Trastuzumab composition generally comprises a mixture of a mainspecies antibody (comprising light and heavy chain sequences of SEQ IDNOS: 13 and 14, respectively), and variant forms thereof, in particularacidic variants (including deamidated variants). Preferably, the amountof such acidic variants in the composition is less than about 25%, orless than about 20%, or less than about 15%. See, U.S. Pat. No.6,339,142. See, also, Harris et al., J. Chromatography, B 752:233-245(2001) concerning forms of Trastuzumab resolvable by cation-exchangechromatography, including Peak A (Asn30 deamidated to Asp in both lightchains); Peak B (Asn55 deamidated to isoAsp in one heavy chain); Peak 1(Asn30 deamidated to Asp in one light chain); Peak 2 (Asn30 deamidatedto Asp in one light chain, and Asp102 isomerized to isoAsp in one heavychain); Peak 3 (main peak form, or main species antibody); Peak 4(Asp102 isomerized to isoAsp in one heavy chain); and Peak C (Asp102succinimide (Asu) in one heavy chain). Such variant forms andcompositions are included in the invention herein.

(iv) 5-FU and Cisplatin

There is no single, standard, globally accepted chemotherapeutic regimenfor advanced gastric cancer, but 5-fluorouracil (5-FU) plus cisplatin iswidely used for this indication. In Phase II studies in patients with noprior chemotherapy. 5-FU+cisplatin produced response rates ofapproximately 40% and median overall survival of 7-10.6 months (Lacave AJ, Baron F J, Anton L M. et al. Ann Oncol 1991; 2:751-754; Rougier P,Ducreux M, Mahjoubi M, et al. Eur J Cancer 1994; 30A:1263-1269;Vanhoefer U, Wagner T, Lutz M. et al. Eur J Cancer 2001:37 Suppl 6:abstract S27.)

(V) Capecitabine

Capecitabine has been extensively tested in patients with advancedgastric cancer. Phase II efficacy results for capecitabine monotherapyshow response rates of 19% and 26% and an overall survival of 8.1 and10.0 months in studies by Koizumi et al 2003 (Koizumi W, Kurihara M.Sasai T, et al. Cancer 1993; 72:658-62; Sakamoto J. Chin K, Kondo K. etal. Anti-Cancer Drugs 2006; 17:2331-6. For capecitabine in combinationwith platinum, there are a number of studies showing response ratesranging from 28% to 65%, time to progression from 5.8 to 9 months, andoverall survival from 10.1 to 12 months (Kang Y, Kang W K, Shin D B, etal. J Clin Oncology 2006:24 Suppl 18:abstract LBA4018; Park Y, Kim B,Ryoo B, et al. Proc Am Soc Clin Oncol 2006:24 Suppl 18:abstract 4079;Kim T W, Kang Y K, Ahn J H, et al. Ann Oncol 2002; 13:1893-8; Park Y H,Kim B S, Ryoo B Y. et al. Br J Cancer 2006; 94:959-63).

III. Selecting Patients for Therapy

Detection of HER2 can be used to select patients for treatment inaccordance with the present invention. Several FDA-approved commercialassays are available to identify HER2-positive cancer patients. Thesemethods include HERCEPTEST® (Dako) and PATHWAY® HER2(immunohistochemistry (IHC) assays) and PathVysion® and HER2 FISHpharmDx™ (FISH assays). Users should refer to the package inserts ofspecific assay kits for information on the validation and performance ofeach assay.

For example, HER2 overexpression may be analyzed by IHC, e.g. using theHERCEPTEST® (Dako). Paraffin embedded tissue sections from a tumorbiopsy may be subjected to the IHC assay and accorded a HER2 proteinstaining intensity criteria as follows:

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for HER2 overexpression assessment maybe characterized as HER2-negative, whereas those tumors with 2+ or 3+scores may be characterized as HER2-positive.

Tumors overexpressing HER2 may be rated by immunohistochemical scorescorresponding to the number of copies of HER2 molecules expressed percell, and can been determined biochemically:

0=0-10,000 copies/cell.

1+=at least about 200,000 copies/cell,

2+=at least about 500,000 copies/cell,

3+=at least about 2,000,000 copies/cell.

Overexpression of HER2 at the 3+ level, which leads toligand-independent activation of the tyrosine kinase (Hudziak et al.,Proc. Natl. Acad. Sci. USA. 84:7159-7163 (1987)), occurs inapproximately 30% of breast cancers, and in these patients, relapse-freesurvival and overall survival are diminished (Slamon et al., Science,244:707-712 (1989), Slamon et al., Science. 235:177-182 (1987)).

The presence of HER2 protein overexpression and gene amplification arehighly correlated, therefore, alternatively, or additionally, the use ofin situ hybridization (ISH), e.g. fluorescent in situ hybridization(FISH), assays to detect gene amplification may also be employed forselection of patients appropriate for treatment in accordance with thepresent invention. FISH assays such as the INFORM™ (sold by Ventana,Arizona) or PathVysion® (Vysis, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of HER2 amplification in the tumor.

Most commonly, HER2-positive status is confirmed using archivalparaffin-embedded tumor tissue, using any of the foregoing methods.

Preferably, HER2-positive patients having a 2+ or 3+ IHC score or whoare FISH or ISH positive are selected for treatment in accordance withthe present invention.

See also U.S. Pat. No. 7,981,418 and Example 11 for alternative assaysfor screening patients for therapy with Pertuzumab.

IV. Pharmaceutical Formulations

Therapeutic formulations of the HER2 antibodies used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), generally inthe form of lyophilized formulations or aqueous solutions. Antibodycrystals are also contemplated (see US Pat Appln 2002/0136719).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimnethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight(less than about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Lyophilized antibody formulations are described in WO 97/04801,expressly incorporated herein by reference.

Lyophilized antibody formulations are described in U.S. Pat. Nos.6,267,958, 6,685,940 and 6,821,515, expressly incorporated herein byreference. The preferred HERCEPTIN® (Trastuzumab) formulation is asterile, white to pale yellow preservative-free lyophilized powder forintravenous (IV) administration, comprising 440 mg Trastuzumab, 400 mgα,α-trehalose dehydrate. 9.9 mg L-histidine-HCl, 6.4 mg L-histidine, and1.8 mg polysorbate 20, USP. Reconstitution of 20 mL of bacteriostaticwater for injection (BWFI), containing 1.1% benzyl alcohol as apreservative, yields a multi-dose solution containing 21 mg/mLTrastuzumab, at pH of approximately 6.0. For further details, see theTrastuzumab prescribing information.

The preferred Pertuzumab formulation for therapeutic use comprises 30mg/mL Pertuzumab in 20 mM histidine acetate, 120 mM sucrose, 0.02%polysorbate 20, at pH 6.0. An alternate Pertuzumab formulation comprises25 mg/mL Pertuzumab, 10 mM histidine-HCl buffer, 240 mM sucrose. 0.02%polysorbate 20. pH 6.0.

The formulation of the placebo used in the clinical trials described inthe Examples is equivalent to Pertuzumab, without the active agent.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Various drugs which can be combined with the HER dimerization inhibitorare described in the Method Section below. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

V. Treatment Methods

In a first aspect of a treatment method herein, a method for extendingprogression free survival (PFS) in a HER2-positive breast cancer patientpopulation by 6 months or more is provided which comprises administeringPertuzumab, Trastuzumab and chemotherapy (e.g. taxane such as Docetaxel)to the patients in the population. Optionally, the patient populationincludes a suitable number of patients (e.g. 200 or more, 300 or more or400 or more patients) so that a statistically significant extension ofPFS in the population can be evaluated.

The phase 111 CLEOPATRA clinical data in Example 3 below show thatmedian PFS assessed by investigators was 12.4 months with placebo plusTrastuzumab plus Docetaxel and 18.5 months with Pertuzumab plusTrastuzumab plus Docetaxel, thus the improvement in median PFS was 6months or more (e.g. 6.1 months) relative to patients not receivingPertuzumab (i.e. patients only receiving Trastuzumab and Docetaxel).

In an additional or alternative embodiment, a method of obtaining anobjective response rate of 80% or more in a HER2-positive breast cancerpatient population is provided which comprises administering Pertuzumab,Trastuzumab and chemotherapy (e.g. taxane, such as Docetaxel) to thepatients in the population.

In a related aspect, a method of combining two HER2 antibodies to treatHER2-positive cancer without increasing cardiac toxicity in aHER2-positive cancer patient population is provided which comprisesadministering Pertuzumab, Trastuzumab. and chemotherapy to the patientsin the population. Optionally, the patient population includes asuitable number of patients (e.g. 200 or more, 300 or more or 400 ormore patients) so that a statistically significant assessment of lack ofcardiac toxicity resulting from the combination can be made. The phaseIII CLEOPATRA clinical data in Example 3 below show that combiningPertuzumab and Trastuzumab does not exacerbate cardiac toxicity. Cardiactoxicity can be monitored for incidence of symptomatic left ventricularsystolic dysfunction (LVSD) or congestive heart failure (CHF), ordecrease in left ventricular ejection fraction (LVEF), e.g. as disclosedin Example 3 below.

Optionally, the breast cancer is metastatic or locally recurrent,unresectable breast cancer, or de novo Stage IV disease, is defined asimmunohistochemistry (IHC) 3+ and/or fluorescence in situ hybridization(FISH) amplification ratio ≥2.0.

Optionally, the patients in the population have not received previoustreatment or have relapsed after adjuvant therapy, have a leftventricular ejection fraction (LVEF) of ≥50% at baseline, and/or have anEastern Cooperative Oncology Group performance status (ECOG PS) of 0 or1.

In an alternative embodiment, the invention concerns a method oftreating early-stage HER2-positive breast cancer comprisingadministering Pertuzumab, Trastuzumab, and chemotherapy to a patientwith the breast cancer, wherein the chemotherapy comprisesanthracycline-based chemotherapy, or carboplatin-based chemotherapy.This aspect of the invention is supported by the clinical data inExample 5. In one embodiment, the chemotherapy comprisesanthracycline-based chemotherapy, e.g. comprising 5-FU, epirubicin, andcyclophosphamide (FEC). In an alternative embodiment, the chemotherapycomprises carboplatin-based chemotherapy, e.g. comprising taxane (e.g.Docetaxel), Carboplatin in addition to HERCEPTIN®/Trastuzumab (e.g. TCHregimen). In one embodiment. Pertuzumab is administered concurrentlywith the anthracycline-based chemotherapy or with the carboplatin-basedchemotherapy, e.g. wherein the Pertuzumab, Trastuzumab and chemotherapyare administered in 3-week cycles with Pertuzumab. Trastuzumab and thechemotherapy being administered on day-1 of each cycle. The data in theexamples herein demonstrates that Pertuzumab administration does notincrease cardiac toxicity relative to the treatment without Pertuzumab(i.e relative to Trastuzumab with anthrycline-based chemotherapy (e.g.FEC) and no Pertuzumab; or relative to Trastuzumab withcarboplatin-based chemotherapy and no Pertuzumab (i.e. TCH). Theearly-stage HER2-positive breast cancer therapy contemplated hereinincludes neoadjuvant and adjuvant therapy.

The invention herein also concerns a method of treating HER2-positivecancer in a patient comprising co-administering a mixture of Pertuzumaband Trastuzumab from the same intravenous bag to the patient. Thisembodiment is applicable to treatment of any HER2-positive cancer,including HER2-positive breast cancer, HER2-positive gastric cancer,HER2-positive metastatic or locally recurrent, unresectable breastcancer, or de novo Stage IV disease, early-stage HER2-positive breastcancer, etc. Optionally, this method further comprises administeringchemotherapy to the patient.

In yet another embodiment, the treatment methods of the presentinvention comprise, consist essentially of, or consist of theadministration of Pertuzumab, Trastuzumab and a chemotherapy, such as aplatin (e.g. cisplatin) and/or a fluoropurimidine (e.g. capecitabineand/or 5-fluorouracil (5-FU)) to treat HER2-positive gastric cancer.

In particular, the treatment methods of the present invention comprise,consist essentially of, or consist of the administration of Pertuzumab,Trastuzumab. and a chemotherapy, such as a platin and/or afluoropurimidine, e.g. cisplatin and/or capecitabine and/or5-fluorouracil (5-FU), to a human patient with metastatic gastriccancer, non-resectable locally advanced gastric cancer, orpost-operatively recurrent gastric cancer. In certain embodiments, thegastric cancer is not amenable to curative therapy.

In an alternative embodiment, a method of treating HER2-positive breastcancer in a patient is provided comprising administering Pertuzumab,Trastuzumab and vinorelbine to the patient. The breast cancer accordingto this embodiment is optionally metastatic or locally advanced.Optionally, the patient has not previously received systemicnon-hormonal anticancer therapy in the metastatic setting.

In another aspect, the invention provides a method of treatingHER2-positive breast cancer in a patient comprising administeringPertuzumab, Trastuzumab, and aromatase inhibitor (e.g. anastrazole orletrozole) to the patient. According to this embodiment, the breastcancer is advanced breast cancer, including hormone receptor-positivebreast cancer such as estrogen receptor (ER)-positive and/orprogesterone receptor (PgR)-positive breast cancer. Optionally, thepatient has not previously received systemic nonhormonal anticancertherapy in the metastatic setting. This treatment method optionallyfurther comprises administering induction chemotherapy (e.g. comprisingtaxane) to the patient.

Therapy in accordance with the present invention extendsprogression-free survival (PFS) and/or overall survival (OS) of thepatient treated.

The antibodies and chemotherapeutic treatments are administered to ahuman patient in accord with known methods. Specific administrationschedules and formulations are described in the examples herein.

According to one embodiment, Pertuzumab is administered at a dose thatproduces a steady-state C_(min) of 20 μg/mL in 90% of patients receivingPertuzumab and Trastuzumab.

According to one particular embodiment of the invention, a Pertuzumab ofapproximately 840 mg (loading dose) is administered, followed by one ormore doses of approximately 420 mg (maintenance dose(s)) of theantibody. The maintenance doses are preferably administered about every3 weeks, for a total of at least two doses, until clinical progressivedisease, or unmanageable toxicity, preferably up to about 6, or 7, or 8,or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17 or moredoses. Longer treatment periods, including more treatment cycles, arealso contemplated.

According to another particular embodiment. Pertuzumab is administeredat a dose of 840 mg for all treatment cycles.

Trastuzumab typically is administered as an intravenous loading dose ofabout 8 mg/kg, followed by the administration of 6 mg/kg doses insubsequent cycles. Trastuzumab is typically administered every 3 weeksuntil clinical progressive disease or unmanageable toxicity, preferablyup to about 17 or more doses.

In a particular embodiment, Trastuzumab is administered as anintravenous (IV) infusion on Day 1 of each treatment cycle untilinvestigator-assessed disease progression or unmanageable toxicity, at aloading dose of 8 mg/kg for Cycle 1 and a dose of 6 mg/kg for subsequentcycles.

In another particular embodiment. Pertuzumab is administered as an IVinfusion on Day 1 of each cycle, for a total of six cycles or untilinvestigator assessed disease progression or unmanageable toxicity,whichever occurs first, either at a loading dose of 840 mg for Cycle 1and a dose of 420 mg for the subsequent cycles, or a loading dose of 840mg for Cycle 1 and a dose of 840 mg for the subsequent cycles.

For treating gastric cancer, Cisplatin 80 mg/m² is typicallyadministered as an IV infusion on Day 1 of each cycle, for a total of atleast six cycles.

For treating gastric cancer, Capecitabine 1000 mg/m² is typicallyadministered orally twice daily, from the evening of Day 1 to themorning of Day 15 of each cycle, for a total of at least six cycles.Capecitabine administration may be prolonged at the discretion of theattending clinician after careful risk-benefit assessment for individualpatients.

Dosages and schedules for chemotherapy used to treat HER2-positivebreast cancer are disclosed in the examples below, but other dosages andschedules are known and contemplated according to the invention herein.

VI. Articles of Manufacture

One embodiment of an article of manufacture herein comprises anintravenous (IV) bag containing a stable mixture of Pertuzumab andTrastuzumab suitable for administration to a cancer patient. Optionally,the mixture is in saline solution; for example comprising about 0.9%NaCl or about 0.45% NaCl. An exemplary IV bag is a polyolefin orpolyvinyl chloride infusion bag, e.g. a 250 mL IV bag. According to oneembodiment of the invention, the mixture includes about 420 mg or about840 mg of Pertuzumab and from about 200 mg to about 1000 mg ofTrastuzumab (e.g. from about 400 mg to about 900 mg of Trastuzumab).

Optionally, the mixture in the IV bag is stable for up to 24 hours at 5°C. or 30° C. Stability of the mixture can be evaluated by one or moreassays selected from the group consisting of: color, appearance andclarity (CAC), concentration and turbidity analysis, particulateanalysis, size exclusion chromatography (SEC), ion-exchangechromatography (IEC), capillary zone electrophoresis (CZE), imagecapillary isoelectric focusing (iCIEF), and potency assay.

In an alternative embodiment, the invention provides an article ofmanufacture comprising a vial with Pertuzumab therein and a packageinsert, wherein the package insert provides the safety data in Table 3or Table 4 and/or the efficacy data in Table 2, Table 5, FIG. 8, or FIG.10. Optionally, the vial is a single-dose vial containing about 420 mgof Pertuzumab. In one embodiment, the vial is provided inside acardboard carton.

In a related aspect, the invention concerns a method of making anarticle of manufacture comprising packaging together a vial withPertuzumab therein and a package insert, wherein the package insertprovides the safety data in Table 3 or Table 4 and/or the efficacy datain Table 2, Table 5, FIG. 8, or FIG. 10.

In a further related aspect, the invention provides a method of ensuringsafe and effective use of Pertuzumab comprising packaging together avial with Pertuzumab therein and a package insert, wherein the packageinsert provides the safety data in Table 3 or Table 4 and/or theefficacy data in Table 2. Table 5. FIG. 8, or FIG. 10.

VII. Deposit of Biological Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

Antibody Designation ATCC No. Deposit Date 4D5 ATCC CRL 10463 May 24,1990 2C4 ATCC HB-12697 Apr. 8, 1999

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

Example 1 Phase IIa Study Evaluating Pertuzumab in Combination withTrastuzumab and Chemotherapy in Patients with HER2-Positive AdvancedGastric Cancer

Despite a sharp worldwide decline in incidence and a reduction inmortality during the second half of the twentieth century, gastriccancer remains the world's second leading cause of cancer mortality,after lung cancer (Parkin. D. Oncogene 23:6329-40 (2004)). The incidenceof gastric cancer varies widely according to geographic region (Kelleyet al. J Clin Epidemiol 56:1-9 (2003); Plummer et al. Epidemiology ofgastric cancer. In: Butlet et al., editors. Mechanisms ofcarcinogenesis: contribution of molecular epidemiology. Lyon: IARCScientific Publications No 157, IARC (2004)). In Japan, Korea. China,and certain countries in Central and South America, the incidence is 20to 95 cases per 100,000 men. In contrast, in the United States, India,and Thailand, the incidence is 4 to 8 cases per 100,000 men. Theincidence in Western Europe ranges from 37 cases per 100,000 men inparts of Italy to 12 per 100.000 men in France. The incidence in womenfollows a similar geographic pattern but is about 50% lower than that inmen. There are clear epidemiological differences between cancerlocalized to the gastric cardia (gastroesophageal junction) and thatlocalized to the rest of the stomach. Cancer of the cardia accounts for39% of gastric cancer cases in white men in the United States but only4% of gastric cancers in men in Japan. For reasons that are not clear,cancer of the gastric cardia and lower esophagus has increased rapidlyin developed countries since the 1970s.

To date, the only potentially curative treatment for gastric cancer issurgery. Survival rates for gastric cancer improved significantly inJapan in recent years as a result of earlier detection and bettersurgical techniques (Inoue et al. Postgrad Med J 81:419-24 (2005)).However, in Western Europe and North America, gastric cancer is oftendiagnosed at a late stage when resection is no longer possible.Consequently, the overall 5-year survival in these populations does notexceed 25% (Ajani, J. The Oncologist 10 Suppl 3:49-58 (2005); Catalanoet al. Clin Rev Oncol/Hematol 54: 209-41 (2005)).

Regardless of their geographic region, patients with unresectabledisease due to locally advanced growth or metastatic spread have a poorprognosis, with overall 5-year survival within the range of 5%-15%(Cunningham et al., Annals of Oncology 16 Suppl 1:i22-3(2005)). Forpatients with unresectable disease at diagnosis and for patients withrecurrent disease after surgery, the main therapeutic option ischemotherapy (National Comprehensive Cancer Network. NCCN clinicalpractice guidelines in oncology. Gastric cancer. Version 1. NationalComprehensive Cancer Network, (2006)). Chemotherapy given withpalliative intent has been shown to be superior to best supportive carein patients with advanced gastric cancer (Wagner et al. J Clin Oncol24:2903-9 (2006)).

Study BO18255 (ToGA) was a randomized, open-label, multicenter,international, comparative Phase III trial designed to evaluate theefficacy and safety of Trastuzumab in combination with chemotherapycompared with chemotherapy alone as first-line therapy in patients withinoperable, locally advanced or recurrent and/or metastaticHER2-positive adenocarcinoma of the stomach or gastroesophagealjunction. The primary objective of the study was to compare overallsurvival for patients treated with Trastuzumab combined withfluoropyrimidine (5-FU or capecitabine) plus cisplatin. The results fromStudy BO18255 demonstrated a significant clinical benefit whenTrastuzumab was used in combination with chemotherapy in patients withgastric cancer. Overall survival, the primary endpoint, wassignificantly improved in the Trastuzumab plus chemotherapy arm comparedwith the chemotherapy alone arm (p=0.0045, log-rank test; hazard ratio,0.74). The median survival time was 13.8 months in the Trastuzumab pluschemotherapy arm and 11.1 months in the chemotherapy alone arm, and therisk of death was decreased by 26% for patients in the Trastuzumab pluschemotherapy arm. All other secondary endpoints demonstrated clinicalsignificance with similar hazard and odds ratios. (See, e.g. Bang etal., Lancet 28; 376(9742):687-97 (2010)).

As a result of this study. Trastuzumab is now indicated, including theEU and United States, in combination with cisplatin plus capecitabine or5-FU, for the treatment of patients with HER2-positive metastaticgastric or gastroesophageal junction adenocarcinoma who have notreceived prior treatment for metastatic disease.

At present, there is no single, standard, globally acceptedchemotherapeutic regimen for advanced gastric cancer. Despite thesuccess of the ToGA trial, there is a great need for providing new andeffective treatment options for this serious condition. In particular,there is a need for novel therapeutic approaches that aim to avoidtreatment-related morbidity and/or to increase survival in gastriccancer patients. Accordingly, this example is a randomized, multicenter,open-label study evaluating two different doses of Pertuzumab inpatients with HER2-positive adenocarcinoma of the stomach orgastroesophageal junction. Patients are randomized in a 1:1 ratio to twotreatment arms. Patients in Arm A receive a Pertuzumab loading dose of840 mg for Cycle 1 and a dose of 420 mg for Cycles 2-6, and patients inArm B receive Pertuzumab 840 mg for all six cycles. Patients in bothtreatment arms receive Trastuzumab, cisplatin, and capecitabine. Studyschema are in FIG. 6. The length of the study is approximately 24 months(4 months for recruitment and 20 months of follow-up after last patientrecruited). The end of study will be when progressive disease hasoccurred in all patients, or all patients have withdrawn or discontinuedfrom the study, whichever is earlier.

Target Population

The trial involves approximately 30 patients.

Patients Must Meet the Following Criteria for Study Entry:

Histologically confirmed adenocarcinoma of the stomach orgastroesophageal junction with inoperable locally advanced or metastaticdisease, not amenable to curative therapy.

Patients with advanced disease who present with a recurrencepost-operatively (when intent of surgery was cure) are also eligible forentry.

Measurable disease, according to the Response Evaluation Criteria inSolid Tumors (RECIST), v1.1, assessed using imaging techniques (computedtomography (CT) or magnetic resonance imaging (MRI)), or non-measurabledisease that can be followed.

HER2-positive tumor defined as either IHC 3+ or IHC 2+ in combinationwith ISH+, as assessed by central laboratory on primary or metastatictumor. ISH positivity is defined as a ratio oft 2.0 for the number ofHER2 gene copies to the number of signals for CEP17.

Availability of formalin-fixed paraffin-embedded (FFPE) tissue with atleast 5 mm of invasive tumor for central confirmation of HER2eligibility is mandatory.

Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1.

Baseline left ventricular ejection fraction (LVEF)≥55% (measured byechocardiogram (ECHO) or multiple-gated acquisition (MUGA) scan).

Life expectancy of at least 3 months.

Male or female.

Age≥18 years.

Signed informed consent.

For women of childbearing potential and male participants with partnersof childbearing potential: agreement to use a highly effectivenon-hormonal form of contraception or two effective forms ofnon-hormonal contraception by the patient and/or partner.

Contraception use must continue for the duration of study treatment andfor at least 6 months after the last dose of study medication.

Patients Who Meet any of the Following Criteria are Excluded from StudyEntry:

Previous chemotherapy for advanced or metastatic disease, except thatprior adjuvant or neoadjuvant therapy is allowed if at least 6 monthshas elapsed between completion of adjuvant or neoadjuvant therapy andenrollment in the study.

Adjuvant or neoadjuvant therapy with a platin is not allowed.

Lack of physical integrity of the upper gastrointestinal tract ormalabsorption syndrome (e.g., patients with partial or total gastrectomycan enter the study, but not those with a jejunostomy probe).

Active (significant or uncontrolled) gastrointestinal bleeding.

Residual relevant toxicity resulting from previous therapy (e.g.,neurological toxicity of ≥Grade≥2 (NCI CTCAE)), with the exception ofalopecia.

Other malignancy within the last 5 years, except for carcinoma in situof the cervix, or basal cell carcinoma.

Any of the following abnormal laboratory tests immediately prior torandomization:

Serum total bilirubin >1.5 times the upper limit of normal (ULN) or, forpatients with known Gilberts syndrome, serum total bilirubin >2×ULN

For patients with no liver and no bone metastases: AST or ALT>2.5×ULN,and alkaline phosphatase (ALP)>2.5×ULN

In patients with liver metastases and no bone metastases: AST orALT>5×ULN, and ALP>2.5×ULN

In patients with liver metastases and bone metastases: AST or ALT>5×ULN,and ALP>10×ULN;

In patients with bone metastases and no liver metastases: AST orALT>2.5×ULN, and ALP>10×ULN

Albumin <25 g/L

Creatinine clearance <60 mL/min

Total white blood cell (WBC) count <2500/μL (<2.5×10⁹/L)

Absolute neutrophil count (ANC)<1500/μL (<1.5×10⁹/L)

Platelets <100,000/μL (<100×10⁹/L)

Serious cardiac illness or medical conditions including but not confinedto:

history of documented heart failure or systolic dysfunction (LVEF<50%);

high-risk uncontrolled arrhythmias, such as atrial tachycardia with aheart rate 2100/min at rest;

significant ventricular arrhythmia (ventricular tachycardia) orhigher-grade AV block (second-degree AV block Type 2 (Mobitz II) orthird-degree AV block):

angina pectoris requiring anti-anginal medication:

clinically significant valvular heart disease;

evidence of transmural infarction on ECG; poorly controlled hypertension(e.g., systolic blood pressure >180 mmHg or diastolic bloodpressure >100 mmHg);

dyspnea at rest due to complications of advanced malignancy or otherdisease, or requirement for supportive oxygen therapy;

treatment with chronic or high-dose corticosteroid therapy;

inhaled steroids and short courses of oral steroids for anti-emesis oras an appetite stimulant are allowed;

clinically significant hearing abnormality; known dihydropyrimidinedehydrogenase deficiency;

history or clinical evidence of brain metastases; serious uncontrolledsystemic intercurrent illness (e.g., infections or poorly controlleddiabetes).

Pregnant or lactating

Women of childbearing potential must have a negative serum pregnancytest within 7 days prior to randomization, irrespective of the method ofcontraception used.

Radiotherapy within 4 weeks prior to start of study treatment, or within2 weeks prior to start of study treatment if palliative radiotherapy isgiven to bone metastastic site peripherally and patient recovers fromany acute toxicity.

Major surgery within 4 weeks prior to start of study treatment, withoutcomplete recovery.

Known active infection with HIV, hepatitis B virus, or hepatitis Cvirus.

Known hypersensitivity to any of the study drugs.

Inability to comply with follow-up testing or procedures, as determinedby the investigator.

Investigational Medical Products: Dose. Route and Regimen

Treatment Cycles are 3 Weeks in Length.

Trastuzumab is administered as an intravenous (IV) infusion on Day 1 ofeach cycle until investigator-assessed disease progression orunmanageable toxicity, at a loading dose of 8 mg/kg for Cycle 1 and adose of 6 mg/kg for subsequent cycles.

Pertuzumab is administered as an IV infusion on Day 1 of each cycle, fora total of six cycles or until investigator assessed disease progressionor unmanageable toxicity, whichever occurs first, as follows for eacharm:

Arm A: Patients receive Pertuzumab at a loading dose of 840 mg for Cycle1 and a dose of 420 mg for Cycles 2-6.Arm B: Patients receive Pertuzumab at 840 mg for Cycles 1-6.

Non-Investigational Medical Products

Treatment Cycles are 3 Weeks in Length.

Cisplatin 80 mg/m² is administered as an IV infusion on Day 1 of eachcycle, for a total of six cycles.

Capecitabine 1000 mg/m² is administered orally twice daily, from theevening of Day 1 to the morning of Day 15 of each cycle, for a total ofsix cycles. (Capecitabine may be prolonged at the discretion of theinvestigator after careful risk-benefit assessment for individualpatients.)

Formulations

Formulation of Pertuzumab

Each lot of the recombinant antibodies produced for clinical purposesmeets viral safety requirements and the United States Pharmacopeia andthe European Pharmacopoeia requirements for sterility. Each lot meetsthe required specifications for identity, purity, and potency.

Pertuzumab is provided as a single-use formulation containing 30 mg/mLPertuzumab formulated in 20-mM L-histidine-acetate (pH 6.0), 120-mMsucrose, and 0.02% polysorbate 20. Each 20-cc vial (14.0 mL solution pervial) contains approximately 420 mg of Pertuzumab.

Formulation of Trastuzumab

Investigational Trastuzumab is supplied as a freeze-dried preparation ata nominal content of 150 mg per vial in most countries (vial size variesby country).

Trastuzumab is formulated in histidine, trehalose, and polysorbate 20.Once reconstituted, each solution contains 21 mg/mL of active drug at apH of approximately 6.0.

Assessments

Efficacy

Investigator-assessed tumor response will be used to summarize bestoverall response at the end of Cycles 3 and 6 for each treatment arm,defined as patients with a complete or partial response as determined byRECIST.

Safely

Safety will be assessed through summaries of adverse events, changes inlaboratory test results, and changes in vital signs.

Pharmacokinetics/Pharmacodynamics

Minimum (trough) serum concentration (C_(min)) for Pertuzumab at Day 43will be assessed. In addition, PK parameters such as CL, Vss, AUC, andhalf-life will be estimated. The evaluation of PK parameters from datacollected up to Day 43 will enable modeling and simulation for anestimated dose that predicts a steady-state trough of ≥22 μg/mL in 90%of patients.

Statistical Analyses

Pharmacokinetic Analyses

Individual and mean serum Pertuzumab concentration-time data will betabulated and plotted by dose level. The serum pharmacokinetics ofPertuzumab will be summarized by estimating total exposure (area underthe curve (AUC)), maximum serum concentration (C_(max)), minimum serumconcentration (C_(min)), time to steady-state C_(max) and C_(min), totalserum clearance, volume of distribution, and elimination half-life(t1/2). Estimates for these parameters will be tabulated and summarizedby descriptive statistics (mean, standard deviation, minimum, andmaximum). Depending on the observed Pertuzumab serum concentration-timedata, a population PK approach may be used to estimate the dose thatwill achieve the PK target concentrations.

Observed C_(max) and C_(min) for Trastuzumab will be tabulated andsummarized by descriptive statistics for each specified PK samplingtimepoint. For all PK analyses, actual times of sample collection(rather than scheduled) will be used.

PK parameters (AUC, C_(max), t1/2) of Pertuzumab will be calculatedusing non-compartmental methods, and the systemic clearance will bederived from the plasma concentrations via standard methods.

Analysis Populations

Intent-to-Treat Population

All randomized patients who receive at least one dose of studymedication will be included in the intent-to-treat population (patientswill be assigned to treatment groups as randomized for analysispurposes).

Safety Population

All patients who received at least one dose of study medication will beincluded in the safety-evaluable population (patients will be assignedto treatment groups as treated.)

Sample Size

The purpose of this study is to assess Cm for Pertuzumab at Day 43 inpatients receiving two different Pertuzumab dose regimens. These datawill then be analyzed using a population PK model to identify a dose ofPertuzumab that will achieve a PK target steady-state troughconcentration of ≥20 μg/mL in approximately 90% of the advanced gastriccancer patients. Analyses using the assumption that Pertuzumab behavessimilarly to Trastuzumab in advanced gastric cancer suggest that withsample size of 15 patients per arm (total of 30 patients in this study),the dose to achieve the desired target concentration can be estimatedwith an acceptable degree of precision (coefficient of variation <15%).

Clinical Results

The clinical results of this phase IIa gastric cancer (GC) study areshown in FIGS. 32-37.

FIG. 32 shows the samples taken and time points.

FIG. 33 shows the demographics of the patient population in the two armsof the GC study, treated with 420 mg (Arm A) or 840 mg (Arm B) ofPertuzumab.

FIG. 34 shows the GC history of the patients in Arms A and B,respectively.

FIG. 35 shows the patient disposition in Arms A and B, respectively.

FIG. 36 shows the Overall Response Rate in Arms A and B, respectively.

Safety Data

Diarrhea was the most common event occurring in 90% of subjects and wastypically Grade 1 and 2 with onset in Cycle 1; no patient discontinuedtherapy because of diarrhea.

Grade ≥3 adverse events (AEs) (>13%) included diarrhea, stomatitis,fatigue/asthenia, decreased appetite, hyponatremia, anemia, andneutropenia. With the exception of neutropenia and hyponatremia (higherin Arm A) and decreased appetite (higher in Arm B), incidence of theseevents was similar in the standard and high-dose Pertuzumab arms.

Asymptomatic change in ejection fraction (EF), neutropenic fever, rash,and drug hypersensitivity reaction were not associated with the higherdose of Pertuzumab.

Serious adverse events (SAEs) occurred in 60% of patients, and incidencewas not associated with high-dose Pertuzumab.

Although more patients withdrew from treatment in Arm B, it is not clearthat this was due to a higher Pertuzumab dose because events peading totreatment discontinuation were not uniform.

Pharmacokinetic (PK) Results

FIG. 37 shows the results of Pertuzumab Day 42 concentration assessmentin gastric cancer (GC) (JOSHUA) versus metastatic breast cancer (MBC)(CLEOPATRA).

Summary of the Results

Pertuzumab trough concentrations are lower in GC compared to MBC.

-   -   Between cycle concentrations (i.e. day 7, 14) are in-line with        expected MBC concentrations as clearance is linear at these        higher concentrations.    -   Trough levels with the 840/420 mg dose are about 37% lower        compared to the CLEOPATRA trial (Example 3), likely due to        non-linear clearance at lower concentrations (incomplete        receptor saturation).    -   840/840 mg dose in GC provides trough concentrations similar to        840/420 mg dose in MBC.    -   Covariates have no impact on PK.

Conclusions

Based on Pertuzumab PK in GC, 840/840 mg dose will be used for thetreatment of gastric cancer. This dose is expected to maintain troughlevels above the target of >20 μg % mL in 90% of patients, and providessimilar trough levels as those observed in MBC.

Example 2 Phase III Study Evaluating Pertuzumab in Combination withTrastuzumab and Chemotherapy in Patients with HER2-Positive AdvancedGastric Cancer

This is a Phase III, randomized, open-label, multicenter clinical studydesigned to assess the efficacy of Pertuzumab in combination withTrastuzumab and chemotherapy in patients with HER2-positive locallyadvanced or metastatic gastric cancer.

Patients in the treatment arm receive Trastuzumab, cisplatin, andcapecitabine and/or 5-fluorouracil. In the other arm, patients are giveneither placebo or Pertuzumab.

Treatment Regimens:

Pertuzumab:

840 mg dose for cycles 1-6.

Trastuzumab

8 mg/kg loading dose followed by 6 mg/kg q3w

Capecitabine

1000 mg/m² bid d1-14 q3w×6

5-Fluorouracil

800 mg/m² day continuous iv infusion d1-5 q3w×6

Cisplatin

800 mg/m² q3w×6

Primary end point:

Overall survival (OS)

Secondary end points:

Progression-free survival (PFS), time to disease progression (TTP),objective response rate (ORR), Clinical Benefit Rate. Duration ofResponse. Qol, safety, pain intensity, analgesic consumption, weightchange, pharmacokinetics.

Mail Patient Selection Criteria

Inclusion Criteria:

-   -   Adenocarcinoma of stomach or GEJ    -   Inoperable locally advanced and/or metastatic disease    -   Measurable (RECIST), or non-measurable evaluable disease    -   HER2-positive tumor: IHC 2+ or 3+ and/or ISH+    -   Adequate organ function and ECOG performance status ≤2    -   Written informed consent

Exclusion Criteria

-   -   Previous adjuvant chemotherapy within 6 months    -   Chemotherapy for advanced disease    -   Congestive heart failure or baseline LVEF<50%    -   Creatinine clearance <60 mL/min

It is expected that the treatment methods described herein, comprisingthe administration of Pertuzumab. Trastuzumab and chemotherapy(s), e.g.cisplatin and capecitabine, will meet the primary end point (OS). Inparticular, it is expected that the treatment methods herein will betherapeutically effective in the gastric cancer patients treated, forexample, by extending survival, including overall survival (OS) and/orprogression-free survival (PFS) and/or time to disease progression (TTP)and/or objective response rate (ORR) relative to treatment withTrastuzumab and chemotherapy only.

Example 3 Results of a Phase III Randomized, Double-Bind,Placebo-Controlled Registration Trial to Evaluate the Efficacy andSafety of Placebo+Trastuzumab+Docetaxel VersusPertuzumab+Trastuzumab+Docetaxel in Patients with Previously UntreatedHER2-Positive Metastatic Breast Cancer (CLEOPATRA)

A protocol for evaluating Pertuzumab in HER2-positive metastatic breastcancer is found at Genentech, Inc., “A study to evaluatepertuzumab+trastuzumab+docetaxel vs. placebo+trastuzumab+docetaxel inpreviously untreated HER2-positive metastatic breast cancer (CLEOPATRA)”(ClinicalTrials.gov Identifier NCT00567190; First received: 12/3/2007;Last updated: Oct. 24, 2012; Last verified 10/2012; Retrieved frominternet 5/8/2013): 1-4 and in US 2009/0137387 as well asWO02009/154651.

This example concerns the clinical data obtained in the randomized,double-blind, placebo-controlled Phase III trial in patients withHER2-positive MBC, who had not received chemotherapy or biologic therapyfor their metastatic disease. Patients were randomized 1:1 to receiveplacebo plus Trastuzumab plus Docetaxel or Pertuzumab plus Trastuzumabplus Docetaxel. The primary endpoint was progression-free survival(PFS), based on tumor assessments. PFS was defined as the time fromrandomization to the first documented radiographic progressive disease(PD) according to response evaluation criteria in solid tumors (RECIST)version 1.0 (Therasse et al. J Natl Cancer Inst 92:205-16 (2000)) ordeath from any cause, if within 18 weeks of the patient's last tumorassessment.

Secondary endpoints included overall survival (OS), PFS by investigatorassessment, objective response rate (ORR), and safety.

Patients:

Eligible patients had centrally confirmed HER2-positive (defined asimmunohistochemistry (IHC) 3+ and/or fluorescence in situ hybridization(FISH) amplification ratio ≥2.0) (Carlson et al. J Natl Compr Canc Netw4 Suppl 3:S1-22 (2006)), locally recurrent, unresectable, or metastaticbreast cancer, or de novo Stage IV disease. Patients were aged ≥18years, had a left ventricular ejection fraction (LVEF) of ≥50% atbaseline (determined by echocardiogram or multiple gated acquisition),and an Eastern Cooperative Oncology Group performance status (ECOG PS)of 0 or 1. Patients may have received one hormonal treatment for MBCprior to randomization, or neoadjuvant or adjuvant systemic breastcancer therapy including Trastuzumab and/or taxanes, provided that theyexperienced a disease-free interval of ≥12 months between completion ofneoadjuvant or adjuvant therapy and diagnosis of metastatic disease.Exclusion criteria included therapy for MBC (other than describedabove); central nervous system metastases; history of exposure to acumulative dose of doxorubicin >360 mg/m² or its equivalent; history ofLVEF decline to <50% during or after prior Trastuzumab therapy; currentuncontrolled hypertension; history of impaired cardiac function;impaired bone marrow, renal, or liver function; current known infectionwith HIV, HBV, or HCV; pregnancy; lactation; and refusal to usenon-hormonal contraception.

Procedures:

Patients received a loading dose of 8 mg/kg Trastuzumab, followed by amaintenance dose of 6 mg/kg every 3 weeks until investigator-assessedradiographic or clinical PD, or unmanageable toxicity. Docetaxel wasadministered every 3 weeks at a starting dose of 75 mg/m², escalating to100 mg/m² if tolerated. Per protocol, the investigator could reduce thedose by 25% to 55 mg/m² or 75 mg/m² (if the patient had been doseescalated) in order to manage tolerability. It was recommended thatpatients received at least 6 cycles of Docetaxel. Pertuzumab or placebowas given at a fixed loading dose of 840 mg, followed by 420 mg every 3weeks until investigator-assessed radiographic or clinical PD, orunmanageable toxicity. In the case of chemotherapy discontinuation dueto cumulative toxicity, antibody therapy was continued until PD,unacceptable toxicity, or withdrawal of consent. All drugs wereadministered intravenously.

Assessments:

PFS was evaluated by standard RECIST-accepted methodology every 9 weeksby each center and by the IRF until IRF-assessed PD. Assessments of LVEFwere performed at baseline, every 9 weeks during the treatment period,at treatment discontinuation, every 6 months in the first year aftertreatment discontinuation, then annually for up to 3 years in thefollow-up period. Laboratory parameters and ECOG status were assessed atevery cycle. Adverse events (AEs) were monitored continuously and gradedaccording to NCI-CTCAE version 3.0. All cardiac events and seriousadverse events (SAEs) that were ongoing at the time of treatmentdiscontinuation were followed until resolution or stabilization up to 1year after the final dose. Cardiac events and treatment-related SAEswith onset post-treatment

Results

Study Population:

A total of 808 patients were enrolled and randomized to receive placeboplus Trastuzumab plus Docetaxel (n=406) or Pertuzumab plus Trastuzumabplus Docetaxel (n=402) (FIG. 7). Baseline characteristics were similarbetween treatment arms (Table 1).

TABLE 1 Baseline Characteristics of the Intent-to-Treat PopulationPlacebo + Pertuzumab + Trastuzumab + Trastuzumab + Docetaxel Docetaxel(n = 406) (n = 402) Sex, n (%) Female 404 (99.5) 402 (100.0) Age, yearsMedian 54.0 54.0 Range 27-89 22-82 Race, n (%) Asian 133 (32.8) 128(31.8) Black 20 (4.9) 10 (2.5) White 235 (57.9) 245 (60.9) Other* 18(4.4) 19 (4.7) Region, n (%) Asia 128 (31.5) 125 (31.1) Europe 152(37.4) 154 (38.3) North America 68 (16.7) 67 (16.7) South America 58(14.3) 56 (13.9) ECOG status, n (%)  0 248 (61.1) 274 (68.2)  1 157(38.7) 125 (31.1) ≥2 1 (0.2) 3 (0.7) Prior treatment status, n (%) Denovo MBC^(†) 214 (52.7) 218 (54.2) Prior adjuvant or neoadjuvant therapy192 (47.3) 184 (45.8) Prior Trastuzumab treatment, n (%) 41 (10.1) 47(11.7) Prior anthracycline treatment, n (%) 164 (40.4) 150 (37.3) Priortaxane treatment, n (%) 94 (23.2) 91 (22.6) Prior hormone treatment^(‡),n (%) 107 (26.4) 114 (28.4) Disease type at screening, n (%)Non-visceral 90 (22.2) 88 (21.9) Visceral 316 (77.8) 314 (78.1) Hormonereceptor status, n (%) ER and/or PgR positive 199 (49.0) 189 (47.0) ERand PgR negative 196 (48.3) 212 (52.7) Unknown 11 (2.7) 1 (0.2) HER2status IHC, n (%) 405 (100) 401 (100) 0 and 1+ 2 (0.5) 4 (1.0) 2+ 32(7.9) 47 (11.7) 3+ 371 (91.6) 350 (87.3) HER2 status FISH, n (%) 387(100) 385 (100) Positive 383 (99.0) 384 (99.7) Negative 4 (1.0) 1 (0.3)*Includes American Indian and Alaska Native ^(†)No prior chemotherapy orbiological therapy ^(‡)In the neoadjuvant/adjuvant or metastatic setting

Progression-Free Survival:

Treatment with Pertuzumab plus Trastuzumab plus Docetaxel significantlyimproved PFS-IRF, stratified by prior treatment status and region,compared with placebo plus Trastuzumab plus Docetaxel (HR=0.62; 95% CI0.51 to 0.75; p<0.0001) (FIG. 8). The median PFS-IVRF was prolonged by6.1 months from 12.4 months with placebo plus Trastuzumab plus Docetaxelto 18.5 months with Pertuzumab plus Trastuzumab plus Docetaxel. The PFSbenefit of Pertuzumab plus Trastuzumab plus Docetaxel treatment wasobserved across all predefined subgroups (FIG. 9).

Assessment of PFS by investigators closely matched PFS-IRF. Median PFSassessed by investigators was 12.4 months with placebo plus Trastuzumabplus Docetaxel and 18.5 months with Pertuzumab plus Trastuzumab plusDocetaxel (HR=0.65; 95% CI 0.54 to 0.78; p<0.0001).

Key Secondary Efficacy Endpoints:

The interim analysis of OS took place when 43% of events (n=165) thatare planned for final OS analysis had occurred. More deaths occurred inthe placebo plus Trastuzumab plus Docetaxel arm (n=96; 23.6%) than inthe Pertuzumab plus Trastuzumab plus Docetaxel arm (n=69; 17.2%) (FIG.10). The HR (0.64; 95% CI 0.47 to 0.88; p=0.0053) for OS did not meetthe O'Brien-Fleming stopping boundary of the Lan-DeMets α-spendingfunction for this interim analysis of survival (HR≤0.603, p≤0.0012), andtherefore, was not statistically significant. However, the data showed astrong trend suggestive of a survival benefit in favor of Pertuzumabplus Trastuzumab plus Docetaxel. At the time of data cut-off, patientsin both treatments arms had been followed for OS for a median of 19.3months (Kaplan-Meier estimate). The ORR was 69.3% and 80.2% in theplacebo plus Trastuzumab plus Docetaxel arm and Pertuzumab plusTrastuzumab plus Docetaxel arm, respectively The difference in responserates between treatment arms was 10.8% (95% CI 4.2 to 17.5; p=0.0011)(Table 2).

TABLE 2 Overall Response Rate Placebo + Pertuzumab + Trastuzumab +Trastuzumab + Docetaxel Docetaxel Patients with IRF-assessed measurable336 (100) 343 (100) disease at baseline, n (%) Objective response rate233 (69.3) 275 (80.2) Complete response rate 14 (4.2) 19 (5.5) Partialresponse rate 219 (65.2) 256 (74.6) Stable disease 70 (20.8) 50 (14.6)Progressive disease 28 (8.3) 13 (3.8) Unable to assess 2 (0.6) 2 (0.6)No response assessment 3 (0.9) 3 (0.9) IRF, independent review facility

Treatment Exposure:

The median number of cycles administered per patient was 15 and 18 withmedian time on treatment estimated to be 11.8 and 18.1 months forplacebo plus Trastuzumab plus Docetaxel and for Pertuzumab plusTrastuzumab plus Docetaxel, respectively. Dose reductions were notpermitted for placebo, Pertuzumab. or Trastuzumab. Patients received amedian of eight cycles of Docetaxel in each arm. Based on the safetypopulation, 61 (15.4%) patients in the placebo plus Trastuzumab plusDocetaxel arm received Docetaxel dose escalation to 100 mg/m² at anycycle compared with 48 (11.8%) patients in the Pertuzumab plusTrastuzumab plus Docetaxel arm. The median Docetaxel dose intensity was24.8 mg/m²/week in the placebo plus Trastuzumab plus Docetaxel arm and24.6 mg/m²/week in the Pertuzumab plus Trastuzumab plus Docetaxel arm.Reasons for permanent discontinuation of all study treatment arepresented in FIG. 7.

Tolerability and Cardiac Safety:

The AE profile during the treatment period was generally balancedbetween treatment arms (Table 3). The incidence of the following AEs(all grades) was >5% higher with Pertuzumab plus Trastuzumab plusDocetaxel: diarrhea, rash, mucosal inflammation, febrile neutropenia,and dry skin.

TABLE 3 Adverse Events (AU Grades) with ≥25% Incidence in Either Arm or≥5% Difference Between Arms and Grade ≥3 Adverse Events with ≥2%Incidence in the Safety Population Placebo + Pertuzumab + Trastuzumab +Trastuzumab + Docetaxel Docetaxel (n = 397) (n = 407) Most common AEs(all grades), n (%) Diarrhea 184 (46.3) 272 (66.8) Alopecia 240 (60.5)248 (60.9) Neutropenia 197 (49.6) 215 (52.8) Nausea 165 (41.6) 172(42.3) Fatigue 146 (36.8) 153 (37.6) Rash 96 (24.2) 137 (33.7) Decreasedappetite 105 (26.4) 119 (29.2) Mucosal inflammation 79 (19.9) 113 (27.8)Asthenia 120 (30.2) 106 (26.0) Edema peripheral 119 (30.0) 94 (23.1)Constipation 99 (24.9) 61 (15.0) Febrile neutropenia 30 (7.6) 56 (13.8)Dry skin 17 (4.3) 43 (10.6) Grade ≥3 AEs with an incidence rate ≥2%, n(%) Neutropenia 182 (45.8) 199 (48.9) Febrile neutropenia 30 (7.6) 56(13.8) Leukopenia 58 (14.6) 50 (12.3) Diarrhea 20 (5.0) 32 (7.9)Neuropathy peripheral 7 (1.8) 11 (2.7) Anemia 14 (3.5) 10 (2.5) Asthenia6 (1.5) 10 (2.5) Fatigue 13 (3.3) 9 (2.2) Granulocytopenia 9 (2.3) 6(1.5) Left ventricular systolic dysfunction 11 (2.8) 5 (1.2) Dyspnea 8(2.0) 4 (1.0) AE, adverse event

The incidence of the following grade ≥3 AEs was >2% higher withPertuzumab plus Trastuzumab plus Docetaxel: neutropenia, febrileneutropenia, and diarrhea (Table 3). The incidence of grade ≥3 febrileneutropenia in patients from Asia was 12% in the placebo plusTrastuzumab plus Docetaxel arm and 26% in the Pertuzumab plusTrastuzumab plus Docetaxel arm; in all other geographical regions theincidence was ≤10% in both arms.

LVSD (all grades) was reported more frequently in the placebo plusTrastuzumab plus Docetaxel arm compared with the Pertuzumab plusTrastuzumab plus Docetaxel arm (8.3% and 4.4%, respectively). Grade ≥3LVSD was reported in 2.8% of patients receiving placebo plus Trastuzumabplus Docetaxel and in 1.2% of patients receiving Pertuzumab plusTrastuzumab plus Docetaxel. Among patients with a post-baseline LVEFassessment, LVEF declines of ≥10 percentage points from baseline to <50%at any stage during treatment were reported in 6.6% and 3.8% of patientsin the placebo plus Trastuzumab plus Docetaxel arm and Pertuzumab plusTrastuzumab plus Docetaxel arm, respectively.

In the safety population, the majority of deaths in both treatment armswere attributed to PD (81 (20.4%) in the placebo arm. 57 (14.0%) in thePertuzumab arm). Deaths due to causes other than PD were generallybalanced and a similar number of patients died due to AEs (10 (2.5%) inthe placebo arm. 8 (2.0%) in the Pertuzumab arm), with infections beingthe most common cause of death due to an AE.

DISCUSSION

These data show that the combination of the anti-HER2 monoclonalantibodies Pertuzumab and Trastuzumab with Docetaxel prolongs PFS inpatients with HER2-positive MBC in the first-line setting. Treatmentwith Pertuzumab plus Trastuzumab plus Docetaxel exceeded expectations byresulting in a statistically significant reduction in PFS risk (HR=0.62)and an improvement in median PFS of 6.1 months.

The combination was well tolerated and Pertuzumab did not increase ratesof symptomatic or asymptomatic cardiac dysfunction. Before the dataherein, it was expected that treatment with two HER2 antibodies wouldexacerbate cardiac toxicity. However, these data show this was not thecase based on the tests herein for evaluating cardiac toxicity:incidence of symptomatic left ventricular systolic dysfunction (LVSD)including congestive heart failure (CHF), decrease in left ventricularejection fraction (LVEF).

Pertuzumab-related AEs, including skin rash, mucosal inflammation, anddry skin, were mostly mild. There was an increased rate of grade ≥3diarrhea and febrile neutropenia with Pertuzumab plus Trastuzumab plusDocetaxel treatment. The control arm in CLEOPATRA had a similar PFS toprevious randomized studies that showed that the combination ofTrastuzumab and Docetaxel in HER2-positive MBC had a median PFS of 11.7months Marty et al. J Clin Oncol 23:4265-74 (2005).

Without being bound by any one theory, these data indicate thattargeting HER2-positive tumors with two anti-HER2 monoclonal antibodieswith complementary mechanisms of action results in a more comprehensiveblockade of HER2 and highlight the clinical importance of preventing theligand-dependent formation HER2 dimers to optimally silence HER2signaling. This study has shown that combined HER2 blockade withTrastuzumab and Pertuzumab improves the outcome of patients withadvanced HER2-positive disease in the first-line setting. These data aresignificant in that they support the first approved use of a HER2dimerization inhibitor for therapy of HER2-positive cancer patients.

Example 4 Article of Manufacture Including Pertuzumab

The phase III clinical data in Example 3 were used in the development ofan article of manufacture comprising a vial (e.g. single-dose vial) withPertuzumab therein and a package insert providing information about thesafety and/or efficacy thereof, as well as a method of making an articleof manufacture comprising packaging together Pertuzumab in a vial (e.g.single-dose vial) and a package insert with prescribing informationregarding Pertuzumab on a package insert as herein below.

Pertuzumab is a sterile, clear to slightly opalescent, colorless to paleyellow liquid for IV infusion. Each single use vial contains 420 mg ofPertuzumab at a concentration of 30 mg/mL in 20 mM L-histidine acetate(pH 6.0), 120 mM sucrose and 0.02% polysorbate 20.

Pertuzumab is supplied in a single-dose vial containing preservativefree liquid concentrate, at a concentration of 30 mg/mL ready forinfusion. Each vial of Pertuzumab drug product contains a total of 420mg Pertuzumab. Store vials in a refrigerator at 2° C. to 8° C. (36° F.to 46° F.) until time of use. Keep vial in the outer carton in order toprotect from light.

Full Prescribing Information Warning: Embryo-Fetal Toxicity

Exposure to PERTUZUMAB can result in embryo-fetal death and birthdefects. Studies in animals have resulted in oligohydramnios, delayedrenal development, and death. Advise patients of these risks and theneed for effective contraception. (5.1, 8.1, 8.6)

1 Indications and Usage

Pertuzumab is indicated for use in combination with Trastuzumab anddocetaxel for the treatment of patients with HER2-positive metastaticbreast cancer who have not received prior anti-HER2 therapy orchemotherapy for metastatic disease.

2 Dosage and Administration

2.1 Recommended Doses and Schedules

The initial dose of Pertuzumab is 840 mg administered as a 60-minuteintravenous infusion, followed every 3 weeks thereafter by a dose of 420mg administered as an intravenous infusion over 30 to 60 minutes. Whenadministered with Pertuzumab, the recommended initial dose ofTrastuzumab is 8 mg/kg administered as a 90-minute intravenous infusion,followed every 3 weeks thereafter by a dose of 6 mg/kg administered asan intravenous infusion over 30 to 90 minutes. When administered withPertuzumab, the recommended initial dose of docetaxel is 75 mg/m2administered as an intravenous infusion. The dose may be escalated to100 mg/m2 administered every 3 weeks if the initial dose is welltolerated.

2.2 Dose Modification

For delayed or missed doses, if the time between two sequentialinfusions is less than 6 weeks, the 420 mg dose of Pertuzumab should beadministered. Do not wait until the next planned dose. If the timebetween two sequential infusions is 6 weeks or more, the initial dose of840 mg Pertuzumab should be re-administered as a 60-minute intravenousinfusion followed every 3 weeks thereafter by a dose of 420 mgadministered as an intravenous infusion over 30 to 60 minutes. Theinfusion rate of Pertuzumab may be slowed or interrupted if the patientdevelops an infusion-associated reaction. The infusion should bediscontinued immediately if the patient experiences a serioushypersensitivity reaction [see Warnings and Precautions (5.2)].

Left Ventricular Ejection Fraction (LVEF):

Withhold Pertuzumab and Trastuzumab dosing for at least 3 weeks foreither:

-   -   a drop in LVEF to less than 40% or    -   LVEF of 40% to 45% with a 10% or greater absolute decrease below        pretreatment values [see Warnings and Precautions (5.2)]

Pertuzumab may be resumed if the LVEF has recovered to greater than 45%or to 40% to 45% associated with less than a 10% absolute decrease belowpretreatment values.

If after a repeat assessment within approximately 3 weeks, the LVEF hasnot improved, or has declined further, discontinuation of Pertuzumab andTrastuzumab should be strongly considered, unless the benefits for theindividual patient are deemed to outweigh the risks [see Warnings andPrecautions (5.2)]. Pertuzumab should be withheld or discontinued ifTrastuzumab treatment is withheld or discontinued. If docetaxel isdiscontinued, treatment with Pertuzumab and Trastuzumab may continue.Dose reductions are not recommended for Pertuzumab. For docetaxel dosemodifications, see docetaxel prescribing information.

2.3 Preparation for Administration

Administer as an intravenous infusion only. Do not administer as anintravenous push or bolus. Do not mix Pertuzumab with other drugs.

Preparation: Prepare the solution for infusion, using aseptic technique,as follows:

-   -   Parenteral drug products should be inspected visually for        particulates and discoloration prior to administration.    -   Withdraw the appropriate volume of Pertuzumab solution from the        vial(s).    -   Dilute into a 250 mL 0.9% sodium chloride PVC or non-PVC        polyolefin infusion bag.    -   Mix diluted solution by gentle inversion. Do not shake.    -   Administer immediately once prepared.    -   If the diluted infusion solution is not used immediately, it can        be stored at 2° C. to 8° C. for up to 24 hours.    -   Dilute with 0.9% Sodium Chloride injection only. Do not use        dextrose (5%) solution.

3 Dosage Forms and Strengths

Pertuzumab 420 mg/14 mL (30 mg/mL) in a single-use vial

4 Contraindications None 5 Warnings and Precautions

5.1 Embryo-Fetal Toxicity

Pertuzumab can cause fetal harm when administered to a pregnant woman.Treatment of pregnant cynomolgus monkeys with Pertuzumab resulted inoligohydramnios, delayed fetal kidney development, and embryo-fetaldeath. If Pertuzumab is administered during pregnancy, or if the patientbecomes pregnant while receiving this drug, the patient should beapprised of the potential hazard to a fetus [see Use in SpecificPopulations (8.1)]. Verify pregnancy status prior to the initiation ofPertuzumab. Advise patients of the risks of embryo-fetal death and birthdefects and the need for contraception during and after treatment.Advise patients to contact their healthcare provider immediately if theysuspect they may be pregnant. If Pertuzumab is administered duringpregnancy or if a patient becomes pregnant while receiving Pertuzumab,immediately report exposure to the Genentech Adverse Event Line at1-888-835-2555. Encourage women who may be exposed during pregnancy toenroll in the MotHER Pregnancy Registry by contacting 1-800-690-6720[see Patient Counseling Information (17)]. Monitor patients who becomepregnant during Pertuzumab therapy for oligohydramnios. Ifoligohydramnios occurs, perform fetal testing that is appropriate forgestational age and consistent with community standards of care. Theefficacy of intravenous hydration in the management of oligohydramniosdue to Pertuzumab exposure is not known.

5.2 Left Ventricular Dysfunction

Decreases in LVEF have been reported with drugs that block HER2activity, including Pertuzumab. In the randomized trial, Pertuzumab incombination with Trastuzumab and docetaxel was not associated withincreases in the incidence of symptomatic left ventricular systolicdysfunction (LVSD) or decreases in LVEF compared with placebo incombination with Trastuzumab and docetaxel [see Clinical Studies(14.1)]. Left ventricular dysfunction occurred in 4.4% of patients inthe Pertuzumab-treated group and 8.30% of patients in theplacebo-treated group. Symptomatic left ventricular systolic dysfunction(congestive heart failure) occurred in 1.0% of patients in thePertuzumab-treated group and 1.8% of patients in the placebo-treatedgroup [see Adverse Reactions (6.1)]. Patients who have received prioranthracyclines or prior radiotherapy to the chest area may be at higherrisk of decreased LVEF. Pertuzumab has not been studied in patients witha pretreatment LVEF value of ≤50%, a prior history of CHF, decreases inLVEF to <50% during prior Trastuzumab therapy, or conditions that couldimpair left ventricular function such as uncontrolled hypertension,recent myocardial infarction, serious cardiac arrhythmia requiringtreatment or a cumulative prior anthracycline exposure to >360 mg/m² ofdoxorubicin or its equivalent. Assess LVEF prior to initiation ofPertuzumab and at regular intervals (e.g., every three months) duringtreatment to ensure that LVEF is within the institution's normal limits.If LVEF is <40%, or is 40% to 45% with a 10% or greater absolutedecrease below the pretreatment value, withhold Pertuzumab andTrastuzumab and repeat LVEF assessment within approximately 3 weeks.Discontinue Pertuzumab and Trastuzumab if the LVEF has not improved orhas declined further, unless the benefits for the individual patientoutweigh the risks [see Dosage and Administration (2.2)].

5.3 Infusion-Associated Reactions, HypersensitivityReactions/Anaphylaxis

Pertuzumab has been associated with infusion and hypersensitivityreactions [see Adverse Reactions (6.1)]. An infusion reaction wasdefined in the randomized trial as any event described ashypersensitivity, anaphylactic reaction, acute infusion reaction orcytokine release syndrome occurring during an infusion or on the sameday as the infusion. The initial dose of Pertuzumab was given the daybefore Trastuzumab and docetaxel to allow for the examination ofPertuzumab-associated reactions. On the first day, when only Pertuzumabwas administered, the overall frequency of infusion reactions was 13.0%in the Pertuzumab-treated group and 9.8% in the placebo-treated group.Less than 1% were grade 3 or 4. The most common infusion reactions(≥1.0%) were pyrexia, chills, fatigue, headache, asthenia,hypersensitivity, and vomiting. During the second cycle when all drugswere administered on the same day, the most common infusion reactions inthe Pertuzumab-treated group (≥1.0%) were fatigue, dysgeusia,hypersensitivity, myalgia, and vomiting. In the randomized trial, theoverall frequency of hypersensitivity/anaphylaxis reactions was 10.8% inthe Pertuzumab-treated group and 9.1% in the placebo-treated group. Theincidence of Grade 3-4 hypersensitivity/anaphylaxis reactions was 2% inthe Pertuzumab-treated group and 2.5% in the placebo-treated groupaccording to National Cancer Institute-Common Terminology Criteria forAdverse Events (NCI-CTCAE) (version 3). Overall, 4 patients inPertuzumab-treated group and 2 patients in the placebo-treated groupexperienced anaphylaxis. Observe patients closely for 60 minutes afterthe first infusion and for 30 minutes after subsequent infusions ofPertuzumab. If a significant infusion-associated reaction occurs, slowor interrupt the infusion and administer appropriate medical therapies.Monitor patients carefully until complete resolution of signs andsymptoms. Consider permanent discontinuation in patients with severeinfusion reactions [see Dosage and Administration (2.2)].

5.4 HER2 Testing

Detection of HER2 protein overexpression is necessary for selection ofpatients appropriate for Pertuzumab therapy because these are the onlypatients studied and for whom benefit has been shown [see Indicationsand Usage (1) and Clinical Studies (14)]. In the randomized trial,patients with breast cancer were required to have evidence of HER2overexpression defined as 3+ IHC by Dako HERCEPTEST® or FISHamplification ratio ≥2.0 by Dako HER2 FISH PHARMDX™ test kit. Onlylimited data were available for patients whose breast cancer waspositive by FISH, but did not demonstrate protein overexpression by IHC.Assessment of HER2 status should be performed by laboratories withdemonstrated proficiency in the specific technology being utilized.Improper assay performance, including use of sub-optimally fixed tissue,failure to utilize specified reagents, deviation from specific assayinstructions, and failure to include appropriate controls for assayvalidation, can lead to unreliable results.

6 Adverse Reactions

The following adverse reactions are discussed in greater detail in othersections of the label:

-   -   Embryo-Fetal Toxicity [see Warnings and Precautions (5.1)]    -   Left Ventricular Dysfunction [see Warnings and Precautions        (5.2)]    -   Infusion-Associated Reactions, Hypersensitivity        Reactions/Anaphylaxis [see Warnings and Precautions (5.3)]

6.1 Clinical Trials Experience

Because clinical trials are conducted under widely varying conditions,adverse reaction rates observed in the clinical trials of a drug cannotbe directly compared to rates in the clinical trials of another drug andmay not reflect the rates observed in clinical practice. In clinicaltrials, Pertuzumab has been evaluated in more than 1400 patients withvarious malignancies and treatment with Pertuzumab was predominantly incombination with other anti-neoplastic agents.

The adverse reactions described in Table 4 were identified in 804patients with HER2-positive metastatic breast cancer treated in therandomized trial. Patients were randomized to receive either Pertuzumabin combination with Trastuzumab and docetaxel or placebo in combinationwith Trastuzumab and docetaxel. The median duration of study treatmentwas 18.1 months for patients in the Pertuzumab-treated group and 11.8months for patients in the placebo-treated group. No dose adjustment waspermitted for Pertuzumab or Trastuzumab. The rates of adverse eventsresulting in permanent discontinuation of all study therapy were 6.1%for patients in the Pertuzumab-treated group and 5.3% for patients inthe placebo-treated group. Adverse events led to discontinuation ofdocetaxel alone in 23.6% of patients in the Pertuzumab-treated group and23.2% of patients in the placebo-treated group. Table 4 reports theadverse reactions that occurred in at least 10% of patients in thePertuzumab-treated group. The most common adverse reactions (>30%) seenwith Pertuzumab in combination with Trastuzumab and docetaxel werediarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheralneuropathy.

The most common NCI-CTCAE (version 3) Grade 3-4 adverse reactions (>2%)were neutropenia, febrile neutropenia, leukopenia, diarrhea, peripheralneuropathy, anemia, asthenia, and fatigue. An increased incidence offebrile neutropenia was observed for Asian patients in both treatmentarms compared with patients of other races and from other geographicregions. Among Asian patients, the incidence of febrile neutropenia washigher in the Pertuzumab-treated group (26%) compared with theplacebo-treated group (12%).

TABLE 4 Summary of Adverse Reactions Occurring in ≥10% of Patients onthe Pertuzumab Treatment Arm in the Randomized Trial Pertuzumab +Placebo + Trastuzumab + Trastuzumab + docetaxel docetaxel n = 407 n =397 Frequency rate % Frequency rate % Body System/Adverse All Grades AllGrades Reactions Grades % 3-4 % Grades % 3-4 % General disorders andadministration site conditions Fatigue 37.6 2.2 36.8 3.3 Asthenia 26.02.5 30.2 1.5 Edema peripheral 23.1 0.5 30.0 0.8 Mucosal inflammation27.8 1.5 19.9 1.0 Pyrexia 18.7 1.2 17.9 0.5 Skin and subcutaneous tissuedisorders Alopecia 60.9 0.0 60.5 0.3 Rash 33.7 0.7 24.2 0.8 Naildisorder 22.9 1.2 22.9 0.3 Pruritus 14.0 0.0 10.1 0.0 Dry skin 10.6 0.04.3 0.0 Gastrointestinal disorders Diarrhea 66.8 7.9 46.3 5.0 Nausea42.3 1.2 41.6 0.5 Vomiting 24.1 1.5 23.9 1.5 Constipation 15.0 0.0 24.91.0 Stomatitis 18.9 0.5 15.4 0.3 Blood and lymphatic system disordersNeutropenia 52.8 48.9 49.6 45.8 Anemia 23.1 2.5 18.9 3.5 Leukopenia 18.212.3 20.4 14.6 Febrile neutropenia* 13.8 13.0 7.6 7.3 Nervous systemdisorders Neuropathy peripheral 32.4 3.2 33.8 2.0 Headache 20.9 1.2 16.90.5 Dysgeusia 18.4 0.0 15.6 0.0 Dizziness 12.5 0.5 12.1 0.0Musculoskeletal and connective tissue disorders Myalgia 22.9 1.0 23.90.8 Arthralgia 15.5 0.2 16.1 0.8 Infections and infestations Upperrespiratory tract 16.7 0.7 13.4 0.0 infection Nasopharyngitis 11.8 0.012.8 0.3 Respiratory, thoracic and mediastinal disorders Dyspnea 14.01.0 15.6 2.0 Metabolism and nutrition disorders Decreased appetite 29.21.7 26.4 1.5 Eye disorders Lacrimation increased 14.0 0.0 13.9 0.0Psychiatric disorders Insomnia 13.3 0.0 13.4 0.0 *In this table thisdenotes an adverse reaction that has been reported in association with afatal outcome

The following clinically relevant adverse reactions were reported in<10% of patients in the Pertuzumab-treated group:

Skin and subcutaneous tissue disorders: Paronychia (7.1% in thePertuzumab-treated group vs. 3.5% in the placebo-treated group);Respiratory, thoracic and mediastinal disorders: Pleural effusion (5.2%in the Pertuzumab-treated group vs. 5.8% in the placebo-treated group);Cardiac disorders: Left ventricular dysfunction (4.4% in thePertuzumab-treated group vs. 8.3% in the placebo-treated group)including symptomatic left ventricular systolic dysfunction (CHF) (1.0%in the Pertuzumab-treated group vs. 1.8% in the placebo-treated group);Immune system disorders: Hypersensitivity (10.1% in thePertuzumab-treated group vs. 8.6% in placebo-treated group).

Adverse Reactions Reported in Patients Receiving Pertuzumab andTrastuzumab after Discontinuation of Docetaxel

In the randomized trial, adverse reactions were reported less frequentlyafter discontinuation of docetaxel treatment. All adverse reactions inthe Pertuzumab and Trastuzumab treatment group occurred in <10% ofpatients with the exception of diarrhea (19.1%), upper respiratory tractinfection (12.8%), rash (11.7%), headache (11.4%), and fatigue (11.1%).

6.2 Immunogenicity

As with all therapeutic proteins, there is the potential for an immuneresponse to Pertuzumab. Patients in the randomized trial were tested atmultiple time-points for antibodies to Pertuzumab. Approximately 2.8%(11/386) of patients in the Pertuzumab-treated group and 6.2% (23/372)of patients in the placebo-treated group tested positive foranti-Pertuzumab antibodies. Of these 34 patients, none experiencedanaphylactic/hypersensitivity reactions that were clearly related to theanti-therapeutic antibodies (ATA). The presence of Pertuzumab in patientserum at the levels expected at the time of ATA sampling can interferewith the ability of this assay to detect anti-Pertuzumab antibodies. Inaddition, the assay may be detecting antibodies to Trastuzumab. As aresult, data may not accurately reflect the true incidence ofanti-Pertuzumab antibody development. Immunogenicity data are highlydependent on the sensitivity and specificity of the test methods used.Additionally, the observed incidence of a positive result in a testmethod may be influenced by several factors, including sample handling,timing of sample collection, drug interference, concomitant medication,and the underlying disease. For these reasons, comparison of theincidence of antibodies to Pertuzumab with the incidence of antibodiesto other products may be misleading.

7 Drug Interactions

No drug-drug interactions were observed between Pertuzumab andTrastuzumab, or between Pertuzumab and docetaxel.

8 Use in Specific Populations

8.1 Pregnancy

Pregnancy Category D

Risk Summary

There are no adequate and well-controlled studies of Pertuzumab inpregnant women. Based on findings in animal studies, Pertuzumab cancause fetal harm when administered to a pregnant woman. The effects ofPertuzumab are likely to be present during all trimesters of pregnancy.

Pertuzumab administered to pregnant cynomolgus monkeys resulted inoligohydramnios, delayed fetal kidney development, and embryo-fetaldeaths at clinically relevant exposures of 2.5 to 20-fold greater thanthe recommended human dose, based on C_(max). If Pertuzumab isadministered during pregnancy, or if a patient becomes pregnant whilereceiving Pertuzumab, the patient should be apprised of the potentialhazard to the fetus. If Pertuzumab is administered during pregnancy orif a patient becomes pregnant while receiving Pertuzumab, immediatelyreport exposure to the Genentech Adverse Event Line at 1-888-835-2555.Encourage women who may be exposed during pregnancy to enroll in theMotHER Pregnancy Registry by contacting 1-800-690-6720 [see PatientCounseling Information (17)].

Animal Data

Reproductive toxicology studies have been conducted in cynomolgusmonkeys. Pregnant monkeys were treated on Gestational Day (GD)19 withloading doses of 30 to 150 mg/kg Pertuzumab, followed by bi-weekly dosesof 10 to 100 mg/kg. These dose levels resulted in clinically relevantexposures of 2.5 to 20-fold greater than the recommended human dose,based on C_(max). Intravenous administration of Pertuzumab from GD19through GD50 (period of organogenesis) was embryotoxic, withdose-dependent increases in embryo-fetal death between GD25 to GD70. Theincidences of embryo-fetal loss were 33, 50, and 85% for dams treatedwith bi-weekly Pertuzumab doses of 10, 30, and 100 mg/kg, respectively(2.5 to 20-fold greater than the recommended human dose, based onC_(max). At Caesarian section on GD100, oligohydramnios, decreasedrelative lung and kidney weights and microscopic evidence of renalhypoplasia consistent with delayed renal development were identified inall Pertuzumab dose groups. Pertuzumab exposure was reported inoffspring from all treated groups, at levels of 29% to 40% of maternalserum levels at GD100.

8.3 Nursing Mothers

It is not known whether Pertuzumab is excreted in human milk, but humanIgG is excreted in human milk. Because many drugs are secreted in humanmilk and because of the potential for serious adverse reactions innursing infants from Pertuzumab, a decision should be made whether todiscontinue nursing, or discontinue drug, taking into account theelimination half-life of Pertuzumab and the importance of the drug tothe mother [See Warnings and Precautions (5.1), Clinical Pharmacology(12.3)].

8.4 Pediatric Use

The safety and effectiveness of Pertuzumab have not been established inpediatric patients.

8.5 Geriatric Use

Of 402 patients who received Pertuzumab in the randomized trial, 60patients (15%) were ≥65 years of age and 5 patients (1%) were ≥75 yearsof age. No overall differences in efficacy and safety of Pertuzumab wereobserved between these patients and younger patients. Based on apopulation pharmacokinetic analysis, no significant difference wasobserved in the pharmacokinetics of Pertuzumab between patients <65years (n=306) and patients ≥65 years (n=175).

8.6 Females of Reproductive Potential

Pertuzumab can cause embryo-fetal harm when administered duringpregnancy. Counsel patients regarding pregnancy prevention and planning.Advise females of reproductive potential to use effective contraceptionwhile receiving Pertuzumab and for 6 months following the last dose ofPertuzumab. If Pertuzumab is administered during pregnancy or if apatient becomes pregnant while receiving Pertuzumab, immediately reportexposure to the Genentech Adverse Event Line at 1-888-835-2555.Encourage women who may be exposed during pregnancy to enroll in theMotHER Pregnancy Registry by contacting 1-800-690-6720 [see PatientCounseling Information (17)].

8.7 Renal Impairment

Dose adjustments of Pertuzumab are not needed in patients with mild(creatinine clearance [CLcr] 60 to 90 mL/min) or moderate (CLcr 30 to 60mL/min) renal impairment. No dose adjustment can be recommended forpatients with severe renal impairment (CLcr less than 30 mL/min) becauseof the limited pharmacokinetic data available [see Clinical Pharmacology(12.3)].

8.8 Hepatic Impairment

No clinical studies have been conducted to evaluate the effect ofhepatic impairment on the pharmacokinetics of Pertuzumab.

10 Overdosage

No drug overdoses have been reported with Pertuzumab to date.

11 Description

Pertuzumab is a recombinant humanized monoclonal antibody that targetsthe extracellular dimerization domain (Subdomain II) of the humanepidermal growth factor receptor 2 protein (HER2). Pertuzumab isproduced by recombinant DNA technology in a mammalian cell (ChineseHamster Ovary) culture containing the antibiotic, gentamicin. Gentamicinis not detectable in the final product. Pertuzumab has an approximatemolecular weight of 148 kDa. Pertuzumab is a sterile, clear to slightlyopalescent, colorless to pale brown liquid for intravenous infusion.Each single use vial contains 420 mg of Pertuzumab at a concentration of30 mg/mL in 20 mM L-histidine acetate (pH 6.0). 120 mM sucrose and 0.02%polysorbate 20.

12 Clinical Pharmacology

12.1 Mechanism of Action

Pertuzumab targets the extracellular dimerization domain (Subdomain II)of the human epidermal growth factor receptor 2 protein (HER2) and,thereby, blocks ligand-dependent heterodimerization of HER2 with otherHER family members, including EGFR. HER3 and HER4. As a result,Pertuzumab inhibits ligand-initiated intracellular signaling through twomajor signal pathways, mitogen-activated protein (MAP) kinase andphosphoinositide 3-kinase (PI3K). Inhibition of these signaling pathwayscan result in cell growth arrest and apoptosis, respectively. Inaddition. Pertuzumab mediates antibody-dependent cell-mediatedcytotoxicity (ADCC). While Pertuzumab alone inhibited the proliferationof human tumor cells, the combination of Pertuzumab and Trastuzumabsignificantly augmented anti-tumor activity in HER2-overexpressingxenograft models.

12.2 Pharmacokinetics

Pertuzumab demonstrated linear pharmacokinetics at a dose range of 2-25mg/kg. Based on a population PK analysis that included 481 patients, themedian clearance (CL) of Pertuzumab was 0.24 L/day and the medianhalf-life was 18 days. With an initial dose of 840 mg followed by amaintenance dose of 420 mg every three weeks thereafter, thesteady-state concentration of Pertuzumab was reached after the firstmaintenance dose. The population PK analysis suggested no PK differencesbased on age, gender, and ethnicity (Japanese vs. non-Japanese).Baseline serum albumin level and lean body weight as covariates onlyexerted a minor influence on PK parameters. Therefore, no doseadjustments based on body weight or baseline albumin level are needed.No drug-drug interactions were observed between Pertuzumab andTrastuzumab. or between Pertuzumab and docetaxel in a sub-study of 37patients in the randomized trial. No dedicated renal impairment trialfor Pertuzumab has been conducted. Based on the results of thepopulation pharmacokinetic analysis, Pertuzumab exposure in patientswith mild (CLcr 60 to 90 mL/min, n=200) and moderate renal impairment(CLcr 30 to 60 mL/min, n=71) were similar to those in patients withnormal renal function (CLcr greater than 90 mL/min, n=200). Norelationship between CLcr and Pertuzumab exposure was observed over therange of observed CLcr (27 to 244 mL/min).

12.3 Cardiac Electrophysiology

The effect of Pertuzumab with an initial dose of 840 mg followed by amaintenance dose of 420 mg every three weeks on QTc interval wasevaluated in a subgroup of 20 patients with HER2-positive breast cancerin the randomized trial. No large changes in the mean QT interval (i.e.,greater than 20 ms) from placebo based on Fridericia correction methodwere detected in the trial. A small increase in the mean QTc interval(i.e., less than 10 ms) cannot be excluded because of the limitations ofthe trial design.

13 Nonclinical Toxicology

13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility

Long-term studies in animals have not been performed to evaluate thecarcinogenic potential of Pertuzumab. Studies have not been performed toevaluate the mutagenic potential of Pertuzumab. No specific fertilitystudies in animals have been performed to evaluate the effect ofPertuzumab. No adverse effects on male and female reproductive organswere observed in repeat-dose toxicity studies of up to six monthsduration in cynomolgus monkeys.

14 Clinical Studies

14.1 Metastatic Breast Cancer

The randomized trial was a multicenter, double-blind, placebo-controlledtrial of 808 patients with HER2-positive metastatic breast cancer.Breast tumor specimens were required to show HER2 overexpression definedas 3+ IHC or FISH amplification ratio ≥2.0 determined at a centrallaboratory. Patients were randomized 1:1 to receive placebo plusTrastuzumab and docetaxel or Pertuzumab plus Trastuzumab and docetaxel.Randomization was stratified by prior treatment (prior or no prioradjuvant/neoadjuvant anti-HER2 therapy or chemotherapy) and geographicregion (Europe, North America, South America. and Asia). Patients withprior adjuvant or neoadjuvant therapy were required to have adisease-free interval of greater than 12 months before trial enrollment.Pertuzumab was given intravenously at an initial dose of 840 mg,followed by 420 mg every 3 weeks thereafter. Trastuzumab was givenintravenously at an initial dose of 8 mg/kg, followed by 6 mg/kg every 3weeks thereafter. Patients were treated with Pertuzumab and Trastuzumabuntil progression of disease, withdrawal of consent, or unacceptabletoxicity. Docetaxel was given as an initial dose of 75 mg/m² byintravenous infusion every 3 weeks for at least 6 cycles. The docetaxeldose could be escalated to 100 mg/m² at the investigator's discretion ifthe initial dose was well tolerated.

At the time of the primary analysis, the mean number of cycles of studytreatment administered was 16.2 in the placebo-treated group and 19.9 inthe Pertuzumab-treated group.

The primary endpoint of the randomized trial was progression-freesurvival (PFS) as assessed by an independent review facility (IRF). PFSwas defined as the time from the date of randomization to the date ofdisease progression or death (from any cause) if the death occurredwithin 18 weeks of the last tumor assessment. Additional endpointsincluded overall survival (OS). PFS (investigator-assessed), objectiveresponse rate (ORR) and duration of response.

Patient demographic and baseline characteristics were balanced betweenthe treatment arms. The median age was 54 (range 22 to 89 years), 59%were White, 32% were Asian, and 4% were Black. All were women with theexception of 2 patients. Seventeen percent of patients were enrolled inNorth America, 14% in South America, 38% in Europe, and 31% in Asia.Tumor prognostic characteristics, including hormone receptor status(positive 48%, negative 50%), presence of visceral disease (78%) andnon-visceral disease only (22%) were similar in the study arms.Approximately half of the patients received prior adjuvant orneoadjuvant anti-HER2 therapy or chemotherapy (placebo 47%, Pertuzumab46%). Among patients with hormone receptor positive tumors. 45% receivedprior adjuvant hormonal therapy and 11% received hormonal therapy formetastatic disease. Eleven percent of patients received prior adjuvantor neoadjuvant Trastuzumab.

The randomized trial demonstrated a statistically significantimprovement in IRF-assessed PFS in the Pertuzumab-treated group comparedwith the placebo-treated group [hazard ratio (HR)=0.62 (95% CI:0.51,0.75), p<0.0001] and an increase in median PFS of 6.1 months (median PFSof 18.5 months in the Pertuzumab-treated group vs. 12.4 months in theplacebo-treated group) (see FIG. 8). The results forinvestigator-assessed PFS were comparable to those observed forIRF-assessed PFS. Consistent results were observed across severalpatient subgroups including age (<65 or ≥65 years), race, geographicregion, prior adjuvant/neoadjuvant anti-HER2 therapy or chemotherapy(yes or no), and prior adjuvant/neoadjuvant Trastuzumab (yes or no). Inthe subgroup of patients with hormone receptor-negative disease (n=408),the hazard ratio was 0.55 (95% CI:0.42, 0.72). In the subgroup ofpatients with hormone receptor-positive disease (n=388), the hazardratio was 0.72 (95% CI:0.55, 0.95). In the subgroup of patients withdisease limited to non-visceral metastasis (n=178), the hazard ratio was0.96 (95% CI:0.61, 1.52).

At the time of the PFS analysis, 165 patients had died. More deathsoccurred in the placebo-treated group (23.6%) compared with thePertuzumab-treated group (17.2%). At the interim OS analysis, theresults were not mature and did not meet the pre-specified stoppingboundary for statistical significance. See Table 5 and FIG. 10.

TABLE 5 Summary of Efficacy from the Randomized Trial Pertuzumab +Placebo + Trastuzumab + Trastuzumab + docetaxel docetaxel HR Parameter n= 402 n = 406 (95% CI) p-value Progression-Free Survival (independentreview) No. of patients with an event 191 (47.5%) 242 (59.6%) 0.62<0.0001 Median months 18.5 12.4 (0.51, 0.75) Overall Survival (interimanalysis) No. of patients with an event 69 (17.2%) 96 (23.6%) 0.640.0053* (0.47, 0.88) Objective Response Rate (ORR) No. of patientsanalyzed Objective response (CR + PR) 343 336 Complete response (CR) 275(80.2%) 233 (69.3%) Partial Response (PR) 19 (5.5%) 14 (4.2%) MedianDuration of Response 256 (74.6%) 219 (65.2%) (months) 20.2 2.5 *The HRand p-value for the interim analysis of Overall Survival did not meetthe pre-defined stopping boundary (HR ≤ 0.603, p ≤ 0.0012).

16 how Supplied/Storage and Handling

16.1 how Supplied

Pertuzumab is supplied as a 420 mg/14 mL (30 mg/mL) single-use vialcontaining preservative-free solution. NDC 50242-145-01. Store vials ina refrigerator at 2° C. to 8° C. (36° F. to 46° F.) until time of use.Keep vial in the outer carton in order to protect from light.

DO NOT FREEZE. DO NOT SHAKE. 17 Patient Counseling Information

Advise pregnant women and females of reproductive potential thatPertuzumab exposure can result in fetal harm, including embryo-fetaldeath or birth defects [see Warnings and Precautions (5.1) and Use inSpecific Populations (8.1)]

Advise females of reproductive potential to use effective contraceptionwhile receiving Pertuzumab and for 6 months following the last dose ofPertuzumab [see Warnings and Precautions (5.1) and Use in SpecialPopulations (8.6)]

Advise nursing mothers treated with Pertuzumab to discontinue nursing ordiscontinue Pertuzumab, taking into account the importance of the drugto the mother [see Use in Specific Populations (8.3)].

Encourage women who are exposed to Pertuzumab during pregnancy to enrollin the MotHER Pregnancy Registry by contacting 1-800-690-6720 [seeWarnings and Precautions (5.1) and Use in Specific Populations (8.1)]

Thus, the comprehensive phase III safety and efficacy data forPertuzumab as in Example 3 provide for the article of manufacture inthis example. This article of manufacture can be used in a method ofensuring safe and effective use of Pertuzumab to treat patients.

Example 5 Early-Stage Breast Cancer Therapy with Pertuzumab

Anthracyclines (generally used in combination with 5-FU andcyclophosphamide) have a central role in the management of breastcancer. Romond et al. NEJM 353(16): 1673-1684 (2005), and Poole et al.NEJM 355 (18): 1851-1852 (2006).

Taxanes are also integral in standard regimens for the treatment ofbreast cancer, used in combination with anthracyclines in a regimenknown as TAC (Martin et al. NEJM 352 (22): 2302-2313 (2005)) or insequence with anthracyclines in a regimen known as AC->T (Romond et al.,supra; Joensuu et al. NEJM 354 (8): 809-820 (2006)).

Carboplatin is both an active and well tolerated chemotherapy agent andthere are studies in breast cancer which show clear efficacy incombination with a taxane and Trastuzumab in a regimen known as TCH(Slamon et al. BCIRG 006. SABS (2007); Robert et al. J. Clin. Oncol. 24:2786-2792 (2006)). However, in metastatic breast cancer, there arenegative data (Forbes et al. BCIRG 007 Proc. Am. Soc. Clin. Oncol.Abstract No. LBA516 (2006)).

A previous neoadjuvant study with Pertuzumab (NeoSphere) evaluated it incombination with Docetaxel and Trastuzumab (Gianni et al. CancerResearch 70 (24) (Suppl. 2) (December 2010)), but not in combinationwith anthracycline-based or carboplatin-based chemotherapy.

The following chemotherapy regimens were evaluated in this example:

FEC Breast cancer therapy consisting of 5-fluorouracil, epirubicin andcyclophosphamide. FEC −>T Sequential chemotherapy, consisting of coursesof FEC chemotherapy followed by courses of Docetaxel. TCH Chemotherapyregimen for HER2-positive breast cancer combination comprising taxane(Docetaxel), Carboplatin, and Trastuzumab (HERCEPTIN ®)

The treatment arms in this study were:

Arm A

Courses of 5-Fluorouracil, Epirubicin and Cyclophosphamide (FEC)followed by courses of Docetaxel (T) (FEC->T) with Trastuzumab andPertuzumab given from the start of the chemotherapy regimen (i.e.concurrently with the anthracycline)

5-Fluorouracil (500 mg/m²), epirubicin (100 mg/m) followed bycyclophosphamide (600 mg/m²) for three cycles, followed by Docetaxel forthree cycles with Trastuzumab (8 mg, kg on day 1 of the first treatmentwith epirubicin and 6 mg/kg every 3 weeks thereafter) and Pertuzumab(840 mg on day 1 of the treatment with FEC with 420 mg every 3 weeksthereafter). The starting dose for Docetaxel is 75 mg/m² for Cycle 4(first Docetaxel cycle) then 100 mg/m² for Cycles 5-6, if no doselimiting toxicity occurs. All drugs will be administered by the IVroute.

OR Arm B

FEC->T with Trastuzumab and Pertuzumab given from the start of thetaxane treatment (i.e. following the anthracycline)

5-Fluorouracil (500 mg/m²), epirubicin (100 mg/m²) followed bycyclophosphamide (600 mg/m²) for three cycles, followed by Docetaxel forthree cycles with Trastuzumab (8 mg/kg on day 1 of the first treatmentwith Docetaxel and 6 mg/kg every 3 weeks thereafter) and Pertuzumab (840mg on day 1 on the first day of Docetaxel with 420 mg every 3 weeksthereafter). The starting dose for Docetaxel is 75 mg/m² for Cycle 4(first Docetaxel cycle) then 100 mg/m² for Cycles 5-6, if no doselimiting toxicity occurs. All drugs will be administered by the IVroute.

OR Arm C

Taxane (Docetaxel), Carboplatin and Trastuzumab (TCH) with Pertuzumab,with both antibodies being given from the start of the chemotherapy.

Carboplatin (AUC6 using Calvert's Formula) followed by Docetaxel on day1 with Trastuzumab (8 mg/kg on day 1 of the first treatment withCarboplatin and Docetaxel and 6 mg/kg every 3 weeks thereafter) andPertuzumab (840 mg on day 1 with 420 mg every 3 weeks thereafter) forsix cycles. The dose for Docetaxel is 75 mg/m² for all cycles. All drugswill be administered by the IV route.

All patients will receive Trastuzumab every three weeks for a total ofone year from the start of treatment (from Cycle 1-17 for patients inArms A and C and Cycle 4-20 for patients in Arm B) whether they receiveadditional chemotherapy or not.

Primary Objective

The primary objective was evaluated when all patients had received sixcycles of neoadjuvant treatment, had their surgery and all necessarysamples taken or were withdrawn from the study whichever is earlier.

Secondary Objectives

To make a preliminary assessment of the activity associated with eachregimen as indicated by the complete pathological response rate.

To evaluate the safety profiles of each treatment regimen, includingpre-operative (neoadjuvant) and post-operative (adjuvant) treatment.

To investigate the overall survival, the time to clinical response,time-to-response, disease free survival and progression free survivalfor each treatment arm.

To investigate the biomarkers that may be associated with primary andsecondary efficacy endpoints in accordance with each treatment arm.

To investigate the rate of breast conservative surgery for all patientswith T2-3 tumors for whom mastectomy was planned at diagnosis.

An overall assessment of the risk and benefit of each regimen will bemade.

Overview of Study Design

This was a Phase II open-label, randomized, multi-center trial toevaluate the tolerability and activity associated with Trastuzumab andPertuzumab when used in addition to anthracycline-based orcarboplatin-based chemotherapy regimens as neoadjuvant therapy inpatients with HER2-positive breast cancer which was early stage and >2cm in diameter or locally advanced or inflammatory (see FIG. 11).

Six cycles of active chemotherapy were administered. However, if it wasconsidered that patients required further therapy post surgery, it wassuggested that those patients who had received FEC->T were given CMF(cyclophosphamide, methotrexate and 5-fluorouracil) and that thosepatients who had received TCH, but who are deemed to require furtherchemotherapy received FEC (5-fluorouracil, epirubicin andcyclophosphamide).

After the completion of surgery (and after the completion ofpost-operative chemotherapy if required), patients received radiotherapyas per local clinical standard and those patients whose tumors wereestrogen-receptor positive received hormone manipulation as per localclinical standard.

In summary, all patients received at least 6 cycles of activechemotherapy and the two antibodies, Pertuzumab and Trastuzumab plussurgery and radiotherapy (as per local standard) plus any hormonemanipulation indicated (as per local standard) and continued to receiveTrastuzumab to one year in total.

Patients whose neoadjuvant study treatment was discontinued prior tosurgery were managed as per local practice. Approximately 28 days afterthe last dose of study medication, patients were asked to perform afinal safety assessment (called Final Visit).

Study Population Overview

Female patients, aged 18 years or more, with early stage HER2-positivebreast cancer whose primary tumors are >2 cm with no metastases.

Inclusion Criteria

-   1. Female patients with locally advanced, inflammatory or early    stage, unilateral and histologically confirmed invasive breast    cancer. The initial breast cancer assessment should be performed by    a physician with experience in surgery for breast cancer. Patients    with inflammatory breast cancer must be able to have a core needle    biopsy.-   2. Primary tumor >2 cm in diameter.-   3. HER2-positive breast cancer confirmed by a central laboratory.    Tumors must be HER2 3+ by IHC or FISH/CISH+(FISH/CISH positivity    mandatory for HER2 2+ tumors).-   4. Availability of FFPE tissue (Buffered Formalin method of fixation    will be accepted) for central confirmation of HER2 eligibility (FFPE    tumor tissue will subsequently be used for assessing status of    biomarkers).-   5. Female patients, age ≥18 years.-   6. Baseline LVEF ≥55% (measured by echocardiography or MUGA).-   7. Performance status ECOG ≤1.-   8. At least 4 weeks since major unrelated surgery, with full    recovery.

Concomitant Medication and Treatment Allowed Therapies

Concomitant treatments are any prescription medications,over-the-counter preparations, herbal remedies or radiotherapy used by apatient in the interval beginning 7 days prior to the patient beingrecruited into the study and continuing through the study.

The following treatments are permitted during the study:

-   1. Acceptable methods of contraception must be used when the female    patient or male partner is not surgical sterilized or does not meet    the study definition of post-menopausal (≥12 months of amenorrhea).-   2. H₁ and H₂ antagonist (e.g. diphenhydramine, cimetidine)-   3. Analgesics (e.g. paracetamol/acetaminophen, meperidine, opioids)-   4. Short term use of corticosteroids to treat or prevent allergic or    infusion reactions-   5. Antiemetics (approved prophylactic serotonin-antagonists,    benzodiazepines, ondansetron etc)-   6. Medication to treat diarrhea (e.g. loperamide)-   7. Colony stimulating factors (e.g. G-CSF)-   8. Estrogen receptor antagonist (e.g. tamoxifen) or aromatase    inhibitors (e.g. anastrazole, exemestane) after completion of    post-operative chemotherapy as per local practice.

Excluded Therapies

The following therapies are excluded during the treatment period of thestudy:

-   9. Anti-cancer therapies other than those administered in this    study, including cytotoxic chemotherapy, radiotherapy. (except for    adjuvant radiotherapy for breast cancer after completion of    chemotherapy or additional adjuvant chemotherapy immediately    post-surgery, if deemed necessary) immunotherapy, and biological    anti-cancer therapy.-   10. Any targeted therapy.-   11. Treatment with steroids except for thyroid hormone replacement    therapy and short term corticosteroid, in order to treat or prevent    allergic or infusion reactions.-   12. High doses of systemic corticosteroids. High dose is considered    as >20 mg of dexamethasone a day (or equivalent) for >7 consecutive    days.-   13. Any investigational agent, except for those used for this study.-   14. Initiation of herbal remedies. Herbal remedies initiated prior    to study entry and continuing during the study are permitted and    must be reported on the appropriate eCRF.-   15. Any oral, injected or implanted hormonal methods of    contraception.

Results

The baseline characteristics of the patients with HER2-positiveearly-stage breast cancer are provided in Table 6 below.

TABLE 6 Baseline Characteristics in the Safety Population FEC + H + P ×3 FEC × 3 → T + → T + H + P × 3 H + P × 3 TCH + P × 6 n = 72 n = 75 n =76 Median age, years (range) 49.0 (27-77) 49.0 (24-75) 50.0 (30-81) ECOGPS 0, n (%) 65 (91.5) 66 (88.0) 67 (88.2) 1, n (%) 6 (8.5) 9 (12.0) 9(11.8) ER and/or PR-positive, n (%) 39 (53.4) 35 (46.7) 40 (51.9) ER andPR-negative, n (%) 34 (46.6) 40 (53.3) 37 (48.1) Disease type, n (%)Operable 53 (72.6) 54 (72.0) 49 (63.6) Locally advanced 15 (20.5) 17(22.7) 24 (31.2) Inflammatory 5 (6.8) 4 (5.3) 4 (5.2) HER2 IHC 0 and 1+,n (%) 1 (1.4) — — 2+, n (%) 5 (6.8) 1 (1.3) 2 (2.6) 3+, n (%) 67 (91.8)74 (98.7) 75 (97.4) HER2 FISH-positive, n (%) 69 (94.5) 69 (92.0) 73(94.8) FISH-negative, n (%) — 1 (1.3) 2 (2.6) Unknown, n (%) 4 (5.5) 5(6.7) 2 (2.6) CBE, clinical breast examination; ECOG PS, EasternCooperative Oncology Group performance status; ER, estrogen receptor;FEC, 5-fluorouracil, epirubicin, cyclophosphamide; FISH, fluorescence insitu hybridisation; H, Trastuzumab; IHC, immunohistochemistry; P,Pertuzumab; PR, progesterone receptor; T, Docetaxel; TCH,Docelaxel/Carboplatin/Trastuzumab

Safety data are shown in FIG. 12 and Tables 7 and 8 below.

TABLE 7 Cardiac Events Overall FEC + H + P × 3 FEC × 3 → T + → T + H + P× 3 H + P × 3 TCH + P × 6 n = 72 n = 75 n = 76 Symptomatic LVSD — 2(2.7) 1 (1.3) (grade ≥3), n (%) LVSD (all grades), n (%) 5 (6.9) 3 (4.0)5 (6.6) LVEF decline ≥10% points 5 (6.9) 5 (6.7) 5 (6.6) from baselineto <50%, n (%) FEC, 5-fluorouracil, epirubicin, cyclophosphamide; H,Trastuzumab; LVEF, left ventricular ejection fraction; LVSD, leftventricular systolic dysfunction; P, Pertuzumab; T, Docetaxel; TCH,Docetaxel/Carboplatin/Trastuzumab

TABLE 8 Ten Most Common Adverse Events During Neoadjuvant TreatmentGrade ≥3 FEC + H + P × 3 FEC × 3 → T + Adverse event, n → T + H + P × 3H + P × 3 TCH + P × 6 (%) n = 72 n = 75 n = 76 Neutropenia 34 (47.2) 32(42.7) 35 (46.1) Febrile neutropenia 13 (18.1) 7 (9.3) 13 (17.1)Leukopenia 14 (19.4) 9 (12.0) 9 (11.8) Diarrhea 3 (4.2) 4 (5.3) 9 (11.8)Anemia 1 (14) 2 (2.7) 13 (17.1) Thrombocytopenia — — 9 (11.8) Vomiting —2 (2.7) 4 (5.3) Fatigue — — 3 (3.9) Alanine — — 3 (3.9) aminotransferaseinc. Drug 2 (2.8) — 2 (2.6) hypersensitivity FEC, 5-fluorouracil,epirubicin, cyclophosphamide; H, Trastuzumab; P, Pertuzumab; T,Docetaxel; TCH, Docetaxel/Carboplatin/Trastuzumab

Efficacy data are provided in FIGS. 13 and 14 as well as Tables 9 and 10below.

TABLE 9 Clinical Response Rate During Neoadjuvant Treatment FEC + H + P× 3 FEC × 3 → T + → T + H + P × 3 H + P × 3 TCH + P × 6 n = 73 n = 75 n= 77 Objective response rate, n (%) 67 (91.8) 71 (94.7) 69 (89.6)Complete response rate 37 (50.7) 21 (28.0) 31 (40.3) Partial responserate 30 (41.1) 50 (66.7) 38 (49.4) Stable disease, n (%) 3 (4.1) 1 (1.3)5 (6.5) Progressive disease, n (%) — 1 (1.3) — No assessment, n (%) 3(4.1) 2 (2.7) 3 (3.9) FEC, 5-fluorouracil, epirubicin, cyclophosphamide;H, Trastuzumab; P, Pertuzumab; T, Docetaxel; TCH,Docetaxel/Carboplatin/Trastuzumab

TABLE 10 Breast Conserving Surgery in Patients for Whom Mastectomy wasPlanned FEC + H + P × 3 FEC × 3 → T + → T + H + P × 3 H + P × 3 TCH + P× 6 n = 46 n = 36 n = 37 Achieved, n (%) 10 (21.7)  6 (16.7) 10 (27.0)(95% CI) (10.9-36.4) (6.4-32.8) (13.8-44.1) Not achieved, n (%) 36(78.3) 30 (83.3) 27 (73.0) CI, confidence interval; FEC, 5-fluorouracil,epirubicin, cyclophosphamide; H, Trastuzumab; P, Pertuzumab; T,Docetaxel; TCH, Docetaxel/Carboplatin/Trastuzumab

CONCLUSIONS

-   -   Results from this study indicate a low incidence of symptomatic        and asymptomatic LVSD across all arms        -   Concurrent administration of Pertuzumab plus Trastuzumab            with epirubicin resulted in similar cardiac tolerability            compared with sequential administration or the            anthracycline-free regimen    -   Neutropenia, febrile neutropenia, leukopenia and diarrhea were        most frequently reported adverse events (grade ≥3) across all        arms    -   Regardless of chemotherapy chosen, the combination of Pertuzumab        with Trastuzumab in the neoadjuvant setting resulted in high        pathological complete response (pCR) rates (57 to 66%)    -   TRYPHAENA supports the use of Pertuzumab and Trastuzumab plus        anthracyline-based or carboplatin-based chemotherapy in the        neoadjuvant and adjuvant settings of early-stage breast cancer.

Example 6 Co-Administration of Pertuzumab and Trastuzumab

In the phase Ill clinical trials above Pertuzumab was administered byintravenous (IV) infusion in saline IV bags to patients withHER2-positive metastatic breast cancer followed by Trastuzumab and thechemotherapeutic agent Docetaxel also using saline IV infusions. The IVinfusion process for Pertuzumab and Trastuzumab takes approximately 60to 90 minutes each with a 30 to 60 minute patient observation periodafter each drug. Due to this treatment regimen per patient, a visit cantake up to 7.5 hours total. As medical payments for both drugs and drugadministration services have been under scrutiny in the recent past,there has been emphasis on business practices to shorten time and toincrease medical resource utilization in clinical and hospital settings.Increased efficiency of patient care, compliance and treatment isexpected by shortening the time patients spend in the clinic for eachcycle of treatment.

As part of the phase Ill Pertuzumab clinical trials, Pertuzumab andTrastuzumab are administered through intravenous (IV) infusion topatients sequentially, i.e. one drug after the other. While Pertuzumabis given as a flat dose (420 mg for maintenance, 840 mg for loading),Trastuzumab is weight based (6 mg/kg for maintenance doses). To increaseconvenience and minimize the in-clinic time for the patients, thefeasibility of co-administering Pertuzumab with Trastuzumab in a single250 mL 0.9% saline polyolefin (PO) or polyvinyl chloride (PVC) IVinfusion bag was assessed. The individual monoclonal antibodies havebeen demonstrated to be stable in infusion bags (PO and/or PVC) over 24hours at 5° C. and 30° C. In this study, the compatibility and stabilityof Pertuzumab (420 mg and 840 mg) mixed with either 420 mg Trastuzumab(6 mg/kg dose for a 70 kg patient) or 720 mg (6 mg/kg for a 120 kgpatient) in IV bags for up to 24 hours at 5° C. or 30° C. was evaluated.The controls (i.e. Pertuzumab alone in an IV bag, Trastuzumab alone inan IV bag) and the monoclonal antibody (mAb) mixture samples wereassessed using the existing Pertuzumab and Trastuzumab analyticalmethods, which include color, appearance and clarity (CAC),concentration and turbidity by UV-spec scan, particulate analysis byHIAC-Royco, size exclusion chromatography (SEC), and ion-exchangechromatography (IEC). Additionally, capillary zone electrophoresis(CZE), image capillary isoelectric focusing (iCIEF), and potency (thePertuzumab anti-proliferation assay only) was utilized to measure theadmixtures containing 1:1 of Pertuzumab:Trastuzumab and their respectivecontrols (420 mg Pertuzumab only and 420 mg Trastuzumab only) only as arepresentative case.

Results showed no observable differences by the above assays in thePertuzumab/Trastuzumab mixtures between the time zero (T0) control andthe sample stored up to 24 hours at either 5° C. or 30° C. Thephysicochemical assays as listed above were able to detect bothmolecules as well as the minor variants in the drug mixture, though someoverlaps of monoclonal antibody species were seen in the chromatograms.Furthermore, the drug mixture tested by the Pertuzumab specificinhibition of cell proliferation assay showed comparable potency beforeand after storage. The results from this study showed the Pertuzumab andTrastuzumab admixtures are physically and chemically stable in an IVinfusion bag for up to 24 hours at 5° C. or 30° C. and can be used forclinical administration if necessary.

Dose I: 840 mg Pertuzumab/Trastuzumab Mixture (420 mg Pertuzumab and 420mg Trastuzumab)

Sample Preparation: All procedures were performed aseptically under alaminar flow hood. PO IV infusion bag samples with three types of drugcombinations were prepared for this study: 1) 420 mg Pertuzumab/420 mgTrastuzumab mixture, 2) 420 mg Pertuzumab alone, and 3) 420 mgTrastuzumab alone. The Pertuzumab and Trastuzumab alone samples servedas controls.

Trastuzumab was reconstituted with 20 mL of bacteriostatic water forinjection (BWFI) and left on lab bench for approximately 15 minutesprior to use. To prepare the Pertuzumab/Trastuzumab sample dose, 14 mLof Pertuzumab (420 mg) was diluted directly into the IV infusion bagthat contained a nominal 250 mL (±25 mL overage) 0.9% saline solution,without removing an equal amount of saline, followed by 20 mL of thereconstituted Trastuzumab (420 mg) using an 18 gauge needle at roomtemperature. The total concentration of the two proteins combined in the250 mL IV bag was expected to be approximately 3 mg/mL. Similarly, thePertuzumab (420 mg) alone IV bag was prepared with 14 mL of the 30 mg/mLdrug product directly diluted into an IV infusion bag. The finalexpected concentration was approximately 1 mg/mL. The Trastuzumab (420mg) alone IV infusion bag was also prepared in the same manner except 20mL of the 21 mg/mL drug product was added into the bag. The finalexpected concentration was approximately 1 mg/mL.

The PO IV bags were manually mixed thoroughly by a gentle back and forthrocking motion several times to ensure homogeneity. After mixing, 10 mLof sample was removed with a syringe from each bag and stored in sterile15 cc falcon tubes to be used as the diluted sample control at time zero(T0). The IV bags were then stored covered in foil at 30° C. for 24hours (T24). Immediately after storage, the remainder of the sample wasremoved with a syringe from each bag and placed into sterile 250 mL PETGcontainers. The T0 and T24 samples were held for up to 24 hours at 5° C.or immediately analyzed by CAC. UV-spec scan (concentration andturbidity), SEC. IEC, CZE, iCIEF, HIAC-Royco, as well as potency. Theproduct quality of the samples was tested by the Pertuzumab andTrastuzumab product specific SEC and IEC methods, while only thePertuzumab specific potency method was performed. The other assaysutilized were non-product specific methods. All assays were qualifiedfor the intended testing in their respective molecules and used withoutfurther method optimization.

Dose II: 1560 mg Pertuzumab/Trastuzumab Mixture (840 mg Pertuzumab and720 mg Trastuzumab)

Sample preparation: The upper range of the mAb co-administration dosewas examined (1560 mg total mixture: 840 mg Pertuzumab and 720 mgTrastuzumab) in PO and PVC IV infusion bag samples. In the event that anincrease in protein aggregation is observed, the propensity of theformation of high molecular weight species (HMWS) would more likelyoccur at the upper dose of 1560 mg total mAb rather than the mixturecontaining 840 mg. To mitigate the risk during in-use conditions at thehigh dose range, both PO and PVC IV infusion bags were studied to ensureno interactions were seen.

Three types of drug combinations (mixture. 840 mg Pertuzumab alone, and720 mg Trastuzumab alone) were prepared and handled similar to the doseI study. The Pertuzumab/Trastuzumab mixture contained 28 mL ofPertuzumab (840 mg) diluted directly into either PO or PVC IV infusionbags followed by 34 mL of the reconstituted Trastuzumab (720 mg) usingan 18 gauge needle at room temperature. The total concentration of thetwo mAbs combined in a single 250 mL IV bag was expected to beapproximately 5 mg/mL. For the controls. Pertuzumab and Trastuzumabalone IV infusion bag samples were prepared and handled similar to thedose I study, except 28 mL of 30 mg/mL Pertuzumab and 34 mL of 21 mg/mLTrastuzumab was directly diluted into each PO or PVC IV infusion bag.The final expected concentration was approximately 3 mg/mL for thePertuzumab (840 mg) and Trastuzumab (720 mg) alone samples. The bagswere stored uncovered at either 5° C. or 30° C. for up to 24 hours. TheT0 and T24 samples were analyzed immediately or held for up to 24 to 48hours at 5° C. by CAC. U V-spec scan (concentration and turbidity). SEC,IEC, and HIAC-Royco.

Details of the types of doses, IV infusion bags, dose & preparation,storage temperatures, and assays are summarized in Table 11.

TABLE 11 IV Bag Type, Dose, Preparation & Study Conditions IV bag typeDilution Storage (approx 250 mL, Total dose, (into approx temperature0.9% NaCl) concentration 250 mL IV bag) (up to 24 hrs) Assays Dose I (n= 1) PO 840 mg, Add 14 mL P (~30 mg/mL) + 30° C. CAC, UV spec scanapprox 3 mg/mL 20 mL T (~21 mg/mL) (concentration, 420 mg^(b), Add 14 mLP (~30 mg/mL) turbidity), SEC, IEC, approx 2 mg/mL CZE, icIEF, HIAC- 420mg^(b), Add 20 mL T (~21 mg/mL) Royco, potency approx 1 mg/mL Dose II (n= 1) PO and PVC^(a) 1560 mg,   Add 28 mL P (~30 mg/mL ) +  5° C. CAC, UVspec scan approx 5 mg/mL 34 mL T (-21 mg/mL) 30° C. (concentration & POand PVC 840 mg^(b), Add 28 mL P (~30 mg/mL) turbidity), SEC, IEC, approx3 mg/mL HIAC-Royco 720 mg^(b), Add 34 mL T (~21 mg/mL) approx 3 mg/mL^(a)n = 2 ^(b)control P = Pertuzumab; T = Trastuzumab

Assays

All samples were held at 5° C. or immediately analyzed. Typically,samples were analyzed within 24 to 48 hours of preparation and storage.The following assays were conducted to ascertain product quality andshort term stability of Pertuzumab/Trastuzumab mixture, Pertuzumabalone, and Trastuzumab alone samples diluted into saline IV infusionbags. Since several assays, i.e. SEC, IEC, CZE, iCIEF, and potency, werenot optimized for quantitative assessment of the mAb mixtures, onlychromatographic or electropherographic overlays of these samples andtheir individual controls before and after storage at 5° C. or 30° C.are shown here. For consistency, no values, e.g. percent peak area, werecalculated for all three sample types from the liquid chromatography andelectrophorectic assays that were performed.

Color, Appearance, and Clarity (CAC)

The color, appearance, and clarity of the samples were determined byvisual inspection under a white fluorescence light with black and whitebackground at room temperature. A 3 cc glass vial was filled with 1 mLof each sample for CAC testing. A negative control (purified water) withthe corresponding sample volume was used for comparison.

UV-Vis Spectrophotometer Scan for Concentration Measurements

The concentration was determined by measurement of the UV-absorbance onan HP8453 spectrophotometer via volumetric sample preparation. Theinstrument was blanked with 0.9% saline. Absorbance at A_(max)(278 nm or279 nm) and 320 nm in a quartz cuvette with 1-cm path length weremeasured for each sample. The absorbance at 320 nm is used to correctfor background light scattering in solution. The concentrationdetermination was calculated by using the absorptivity of 1.50 (mg/mL)⁻¹cm⁻¹ for both Pertuzumab and Trastuzumab molecules.

${{Protein}\mspace{14mu} {Concentration}\mspace{14mu} \left( {{mg}\text{/}{mL}} \right)} = {\frac{A_{\max} - A_{320}}{1.50} \times {Dilution}\mspace{14mu} {Factor} \times \frac{1}{{cuvette}\mspace{14mu} {pathlength}}}$

Size Exclusion Chromatography (SEC: Pertuzumab Specific and TrastuzumabSpecific)

Each sample was injected into a TOSOHAAS® column G3000 SWXL, 7.8×300 mmat ambient temperature on an AGILENT 1100® HPLC. The eluted peaks weremonitored at 280 nm. Chromatographic integrations were analyzed by theCHROMELEON® software. The autosampler temperature was held at 2-8° C.throughout the run and mobile phases used were 0.2M potassium phosphate.0.25 mM potassium chloride, pH 6.2 and 100 mM potassium phosphate. pH6.8 for Pertuzumab-assay and Trastuzumab-assay, respectively. Therecommended injection load as specified by the test procedure was 200 μgwith an injection volume of 20 μL. The diluted 420 mg sample wasinjected at a load less than the recommended amount due to the lowconcentration of the protein after dilution in the IV bags. The maximuminjection volume of the HPLC sample loop was 100 μL, which limits thevolume that is able to be injected at one time. As a result, theinjection volumes were modified to 100 μL at 160 μg protein for thePertuzumab alone and Trastuzumab alone samples (420 mg dose group) and73 μL at 200 μg protein for the Pertuzumab/Trastuzumab mixture (840 mgdose group). Modification in the injection volumes have been utilized inprevious IV bag studies and are necessary when handling lowconcentration samples.

Ion-Exchange Chromatography (IEC)

The analysis of carboxypeptidase B (CpB)-digested Pertuzumab andTrastuzumab for charge heterogeneity was employed by IEC for eachsample. For the Pertuzumab specific IEC, samples were either tested withregular IEC (“Pertuzumab-regular IEC”) or a modified “fast” version ofIEC (“Pertuzumab-IEC-fast”) for high throughput, method for the purposeof these experiments. The IEC assays utilized the a DIONEX® WCX weakcation exchange column equilibrated with solvent A (20 mM MES, 1 mMNa₂EDTA pH 6.00) and solvent B (250 mM sodium chloride in solvent A)monitored at 280 nm for Pertuzumab-regular IEC and Pertuzumab-IEC-fast,whereas solvent A (10 mM sodium phosphate, pH 7.5) and solvent B (100 mMsodium chloride in Solvent A) monitored at 214 nm was used forTrastuzumab on an AGILENT 1100® HPLC. The peaks were eluted at a flowrate of 0.8 mL/min with an increasing gradient of 18%-100% solvent Bover 35 minutes and 90 minutes for Pertuzumab-regular-IEC andPertuzumab-IEC-fast respectively, and 15%-100% solvent B over 55 minutesfor Trastuzumab-IEC. Column temperatures were maintained at either 34°C. or 42° C. and ambient for Pertuzumab-regular-IEC orPertuzumab-fast-IEC and Trastuzumab-IEC, respectively, while the autosampler temperature was held at 2-8° C. throughout the run.

HIAC-ROYCO™ Light Obscuration for Sub-Visible Particles

Particulate counts in the diluted drug product were carried out usingthe HIAC-ROYCO™ Liquid Particulate Counting System model 9703. Averagecumulative numbers of particles at ≥10 μm and ≥25 μm per milliliter weretabulated in each sample using PHARMSPEC v2.0™. The test procedure wasmodified for a small-volume method, utilizing either four 1 mL readingsor four 0.4 mL readings per a test session while discarding the firstreading of each sample. The HIAC-ROYCO™ samples were degassed undervacuum for approximately 10-15 minutes each. The size below 10 μm wasnot collected for this sample set.

UV-Vis Spectrophotometer Scan for Turbidity Measurements

The optical density of the samples from the IV bag (1 mg/mL or 3 mg/mL)was measured in a quartz cuvette with a 1-cm path length on a HP8453spectrophotometer. The sample readings were blanked against purifiedwater. The absorbance measurements were recorded at 340 nm, 345 nm, 350nm, 355 nm, and 360 nm and the turbidity was expressed as an average ofthese wavelengths.

Capillary Zone Electrophoresis, CZE

CZE was performed using a PROTEOMELAB PA800™ capillary electrophoresissystem (Beckman Coulter) with neutral-coated capillary (50 μm×50 cm).The buffer consisted of 40 mM ε-amino caproic acid/acetic acid. pH 4.5,0.2% hydroxypropyl methyl cellulose (HPMC). Samples were diluted to 0.5mg/mL in water and injected into the capillary at 1 psi for 10 seconds.Separation was performed using a voltage of 30 kV for 15 minutes, andthe species were detected by UV at 214 nm.

CE-SDS-LIF, Reduced and Non-Reduced

Each sample was derivatized with 5 carboxytetramethylrhodaminesuccinimidyl ester, a fluorescent dye. After removing the free dyethrough gel filtration (using NAP-5 columns), non-reduced samples wereprepared by adding 40 mM iodoacetamide and heated at 70° C. for 5minutes. For the analysis of the reduced samples, the derivatizedsamples were mixed with SDS to a final concentration of 1% (v/v) and 10mL of a solution containing 1 M DTT, and heated at 70° C. for 20minutes. The prepared samples were analyzed on a Beckman CoulterProteomeLab PA800 system using a 50 mm I.D. 31.2 cm fused silicacapillary maintained at 20° C. throughout the analysis. Samples wereintroduced into the capillary by electrokinetic injection at 10 kV for40 seconds. The separation was conducted at a constant voltage of 15 kVin the reversed polarity (negative to positive) mode using CE-SDSrunning buffer as the sieving medium. An argon ion laser operating at488 nm was used for fluorescence excitation with the resulting emissionsignal monitored at 560 nm.

iCIEF

The distribution of charge variants of the Pertuzumab/Trastuzumabmixture. Pertuzumab alone, and Trastuzumab alone was assessed by iCIEFusing an iCE280™ analyzer (Convergent Bioscience) with a fluorocarboncoated capillary cartridge (100 μm×5 cm). The ampholyte solutionconsisted of a mixture of 0.35% methyl cellulose (MC), 0.47% Pharmalyte3-10 carrier ampholytes. 2.66% Pharmalyte 8-10.5 carrier ampholytes, and0.20% pI markers 7.05 and 9.77 in purified water. The anolyte was 80 mMphosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in0.10% methylcellulose. Samples were diluted in purified water and CpBwas added to each diluted sample at an enzyme to substrate ratio of1:100 followed by incubation at 37° C. for 20 minutes. The CpB treatedsamples were mixed with the ampholyte solution and then focused byintroducing a potential of 1500 V for one minute, followed by apotential of 3000 V for 10 minutes. An image of the focused Pertuzumabcharge variants was obtained by passing 280 nm ultraviolet light throughthe capillary and into the lens of a charge coupled device digitalcamera. This image was then analyzed to determine the distribution ofthe various charge variants.

Anti-Proliferation Potency Assay

This test procedure is based on the ability of Pertuzumab to inhibit theproliferation of MDA MB 175 VII human breast carcinoma cells. Briefly,cells were seeded in 96-well tissue culture microtiter plates andincubated overnight at 37° C. under 5% CO₂ to allow cell attachment. Thefollowing day, the culture medium was removed and serial dilutions ofeach standard, controls, and sample(s) were added to the plates. Theplates were then incubated for fours days at 37° C. under 5% CO₂ and therelative number of viable cells was quantified indirectly using a redoxdye, ALAMARBLUE® according to the manufacturer's protocol. Each samplewas assayed in triplicate and the changes in color as measured byfluorescence were directly proportional to the number living cells inthe culture. The absorbance of each well was then measured on afluorescence 96-well plate reader. The results, expressed in relativefluorescence units (RFU), were plotted against the antibodyconcentration. No quantitative measurements were made, or possible,since there was no Pertuzumab/Trastuzumab mixture reference available.Therefore, the results are comparisons of the dose response curves only.

Results and Discussion Dose I: 840 mg Total Pertuzumab/TrastuzumabMixture (420 mg Pertuzumab and 420 mg Trastuzumab)

The product quality of the total 840 mg Pertuzumab/Trastuzumab mixture(420 mg Pertuzumab and 420 mg Trastuzumab), Pertuzumab alone (420 mg),and Trastuzumab alone (420 mg) in IV infusion bags (n=1) before andafter storage at 30° C. for up to 24 hours was assessed by CAC,concentration measurements by UV-spec scan, turbidity, and HIAC Royco(Table 12). The Pertuzumab and Trastuzumab alone IV infusion bags areconsidered controls that were also prepared to assess the ability of theassay to pick up the appropriate product attributes.

TABLE 12 Dose I 840 mg: Stability data for Pertuzumab/Trastuzumabmixture, Pertuzumab, or Trastuzumab in 0.9% saline PO IV infusion bags(n = 1) Light Obscuration total total IV bag Amount Timepoint TempCAC^(a) Conc. Turbidity particles particles Sample type mg Hour(s) ° C.liquid mg/mL AU ≥10 um/mL ≥25 um/mL pertuzumab/ PO 840 0 30 CL, CO 2.70.016 1 0 trastuzumab 840 24 30 CL, CO 2.7 0.016 6 0 mixture pertuzumabPO 420 0 30 CL, CO 1.4 0.012 3 0 420 24 30 CL, CO 1.4 0.011 4 0trastuzumab PO 420 0 30 CL, CO 1.5 0.012 1 0 420 24 30 CL, CO 1.5 0.0116 0 saline only — — — — — — 2 I ^(a)Color, Appearance and Clarity: CL =clear; SOPL = slightly opalescent; CO = colorless. RT = room temperature^(a)Color, appearance, and clarity: CL = clear; SOPL = slightlyopalescent, CO = colorless

After storage, the Pertuzumab/Trastuzumab mixture, Pertuzumab alone, andTrastuzumab alone samples appeared as a clear and colorless liquid withno visible particles as observed by CAC. The concentration and turbiditymeasurements showed no measurable changes in any of the three sampletypes after 24 hours at 30° C. Particulate analysis by HIAC Roycodetected no more than 6 particles greater than or equal to 10 μm sizeand no particles greater than 25 μm size for Pertuzumab/Trastuzumabmixture, Pertuzumab alone, or Trastuzumab alone samples post storage.These results are comparable to the 0.9% saline only solution. The lackof visible precipitation or particulates indicates that the admixtureand the controls are sufficiently stable upon dilution in the 0.9%saline IV infusion bags. The Pertuzumab/Trastuzumab mixture diluted insaline were run on SEC, both Pertuzumab and Trastuzumab specificmethods, and showed comparable peak profiles between T0 and T24 (FIGS.15 and 16). No increases were observed in the high molecular weightspecies (HMWS) and low molecular weight species (LMWS). Similarly, nochanges were observed in the main peak in any sample. The main peak andthe peak area of the HMWS and LMWS overlay and cannot be distinguishedin the Pertuzumab/Trastuzumab mixture due the size similarity betweenPertuzumab and Trastuzumab (molecular weight approximately 150 kD).Furthermore, comparison of T0 and T24 for both the Pertuzumab andTrastuzumab alone sample showed no observable changes in peak area orprofile as detected by the two SEC methods listed above.

Two product specific methods for Pertuzumab or Trastuzumab IEC wasutilized to analyze the Pertuzumab/Trastuzumab mixture (FIGS. 17 and18). In the cation-exchange chromatography assays, each moleculetypically contain three distinct areas that are eluted based on relativecharge, with the early eluting acidic variants, followed by the mainpeak, and lastly the late eluting basic variants. In the Pertuzumab andTrastuzumab alone chromatograms, the profile exhibiting the acidicvariants, main peak, and the basic variants was observed and deemedcomparable between the starting material and post storage at 30° C.These results are also consistent with prior studies conducted in salineIV infusion bags for the either Pertuzumab or Trastuzumab alone. For thePertuzumab/Trastuzumab mixture chromatogram, the Pertuzumab peaks elutefirst followed by the Trastuzumab peaks. Due to the nature ofcation-exchange separation and the net charge difference betweenPertuzumab (˜pI 8.7) and Trastuzumab (˜pI 8.9), two main peaks, or majorcharged species, are observed in the Pertuzumab/Trastuzumab mixture. Incontrast, the SEC assay separates based on the hydrodynamic size of themolecule and show only one main peak due to the size similarity betweenPertuzumab and Trastuzumab. The charged regions of each molecule appearto overlap with each other in the Pertuzumab/Trastuzumab mixture.Specifically, the Pertuzumab basic variants expected to elute atapproximately 32 minutes and at 35 minutes appear to overlap with themain peak of Trastuzumab (FIGS. 17 and 18). Furthermore, the acidicvariants of Trastuzumab expected to elute before the Trastuzumab mainpeak co-elute with the Pertuzumab basic variants and main peak. Despitethe overlapping peak regions, the Pertuzumab/Trastuzumab mixtureexhibited comparable chromatographic peak profiles before and afterstorage in IV saline bags for 24 hours at 30° C.

The Pertuzumab/Trastuzumab mixture, Pertuzumab alone, and Trastuzumabalone samples were also assayed on CE-SDS LIF under non-reducedconditions after storage for 24 hours at 30° C. ThePertuzumab/Trastuzumab mixture showed consistent peak profiles with noobservable changes after storage compared to the starting material(FIGS. 19 and 20). A very slight baseline level variation attributed tonoise is also observed and does not impact peak area. Similar to SEC,the non-reduced Pertuzumab/Trastuzumab mixture showed only onesuperimposed monomer constituting both the Pertuzumab and Trastuzumabmain species. The Pertuzumab and Trastuzumab alone samples showed nochanges at T0 compared to T24. However, individual molecular attributes,e.g. fragment peak level and species, between Pertuzumab/Trastuzumabmixture, Pertuzumab alone, and Trastuzumab alone was observed asexpected.

Two major peaks known as the light chain (LC) and heavy chain (HC) aredetected at 17 and 21.5 minutes, respectively, whenPertuzumab/Trastuzumab mixture. Pertuzumab alone, and Trastuzumab alonewas run on CE-SDS LIF reduced with DTT (FIG. 20). No increase infragmentation or concomitant decrease in the LC and HC was seen poststorage at 30° C. for the Pertuzumab/Trastuzumab mixture. Furthermore,no detectable peak profile differences were noticed in the Pertuzumaband Trastuzumab alone samples post storage.

The charge separation assays CZE and iCIEF show comparable peak profilesfor the Pertuzumab/Trastuzumab mixture after storage at 30° C. (FIGS. 21and 22). The Pertuzumab and Trastuzumab alone when compared to theirrespective T0 also showed consistent peak profiles with no changes afterstorage. Furthermore, the presence of various minor species was alsoobserved, although no new peaks were detected upon dilution in the IVbag saline solution. As seen in the charge based IEC assay, two mainpeaks flanked by smaller overlapping peaks can be detected and wasattributed to the difference in the molecular pI.

The potency results based on comparison of the dose response curveshowed no impact on the potency of the Pertuzumab/Trastuzumab mixturestored at 30° C. for 24 hours compared to its corresponding T0 doseresponse curve (FIG. 23). The Trastuzumab alone showed little activityin the Pertuzumab potency assay. The Pertuzumab/Trastuzumab mixture doseresponse curve compared to the dose response curve of Pertuzumab orTrastuzumab alone showed that lower doses of the Pertuzumab/Trastuzumabmixture were needed to inhibit the growth of cells as compared toPertuzumab alone, suggesting there may be an additive or synergisticeffect on the inhibition of cell proliferation for the mixture.

Dose II: 1560 mg Total Pertuzumab/Trastuzumab Mixture (840 mg Pertuzumaband 720 mg Trastuzumab)

In addition to the dose I study at 840 mg total mAb, a higher dose of1560 mg mixture (840 mg Pertuzumab and 720 mg Trastuzumab), and theirindividual drug product controls (840 mg Pertuzumab alone and 720 mgTrastuzumab alone) was selected to investigate the impact of dilutingthese three mAb types in PO or PVC IV infusion bags at 5° C. or 30° C.for up to 24 hours. The product quality of these IV infusion bags beforeand after storage was assessed by CAC, UV-spec scan (concentration andturbidity), and HIAC-ROYCO™ are summarized in Table 13 and SEC and IECare shown in FIGS. 24-27.

TABLE 13 Dose II 1560 mg: Stability data for Pertuzumab/Trastuzumabmixture, Pertuzumab, or Trastuzumab in 0.9% saline PO IV infusion bags(n = 1 for control; n = 2 for mixture) Light Obscuration total total IVbag Amount Temp Timepoint CAC^(a) Conc. Turbidity particles particlesSample type mg ° C. Hour(s) liquid mg/mL AU ≥10 um/mL ≥25 um/mLpertuzumab/ PO 1560 5 0 CL, CO 4.9 0.013 20 1 trastuzumab 24 CL, CO 4.70.016 15 1 mixture 1560 30 0 CL, CO 5.0 0.014 8 0 24 CL, CO 4.9 0.018 180 pertuzumab PO 840 5 0 CL, CO 2.9 0.007 1 0 24 CL, CO 2.9 0.005 6 0 84030 0 CL, CO 2.8 0.006 6 0 24 CL, CO 2.8 0.004 9 0 trastuzumab PO 720 5 0CL, CO 2.6 0.004 4 0 24 CL, CO 2.6 0.005 13 0 720 30 0 CL, CO 2.5 0.00719 0 24 CL, CO 2.5 0.004 14 0 pertuzumab/ PVC 1560 5 0 CL, CO 4.9 0.01618 0 trastuzumab 24 CL, CO 4.7 0.015 18 0 mixture 1560 30 0 CL, CO 4.80.016 24 0 24 CL, CO 4.8 0.012 17 0 pertuzumab PVC 840 5 0 CL, CO 2.90.006 13 0 24 CL, CO 2.7 0.004 10 0 840 30 0 CL, CO 2.8 0.006 6 0 24 CL,CO 2.8 0.006 11 0 trastuzumab PVC 720 5 0 CL, CO 2.5 0.007 7 o 24 CL, CO2.5 0.004 9 0 720 30 0 CL, CO 2.5 0.003 18 0 24 CL, CO 2.5 0.005 19 0^(a)Color, Appearance and Clarity: CL = clear; SOPL = slightlyopalescent, CO = colorless.

Two PO or PVC IV infusion bags each were prepared for thePertuzumab/Trastuzumab mixture condition while only one IV infusion bagwas prepared for the Pertuzumab and Trastuzumab alone samples.

Particulates from these bags were determined by visual observation,turbidity, and HIAC-Royco measurements. All samples appeared clear andcolorless after storage at 5° C. or 30° C. for up to 24 hours. Novisible particulate matter was observed and there was no significantchange in the turbidity post storage. For the Pertuzumab/Trastuzumabmixture. Pertuzumab alone, and Trastuzumab alone, the HIAC-Royco showedcomparable particle values before and after storage, with zero to 10particles increase per milliliter at ≥10 μm and zero particle increaseper milliliter at ≥25 μm for both PO and PVC IV infusion bags stored ateither 5° C. or 30° C. Similarly, the Pertuzumab and Trastuzumab alonesamples also exhibited no significant particle differences before andafter storage in PO or PVC IV infusion bags. For all three sample types,the UV-spec scan showed no changes beyond normal assay variability inprotein concentration, indicating the absence of protein adsorption orprecipitation in the IV infusion bags between T0 and T24 hours at 5° C.or 30° C. storage.

The Pertuzumab/Trastuzumab mixture, Pertuzumab alone, and Trastuzumabalone samples were analyzed using Pertuzumab or Trastuzumab specific SECand IEC methods to assess their physical and chemical stability,respectively, as previously described. For the Pertuzumab/Trastuzumabmixture, no changes in SEC were observed in the chromatographic profilesbetween the T0 and the T24 hour samples at 5° C. or 30° C. in either POor PVC IV infusion bags (FIGS. 24 and 25), similar to the 840 mg mixturedose I results. In addition, no increase or decrease in the highmolecular weight species (HMWS), main peak, and low molecular weightspecies (LMWS) was observed, which indicates a stable dosing solution atthe upper ranges of protein content in 0.9%/o saline. Likewise,Pertuzumab alone and Trastuzumab alone samples also showed no changesafter storage in the IV infusion bags.

IEC analysis, using both the Pertuzumab or Trastuzumab specific methods,of the Pertuzumab Trastuzumab mixture was used to assess chemicalstability and showed comparable charge variant peak profiles with noobserved changes relative to the initial time point after exposure to 5°C. or 30° C. in the PO or PVC IV infusion bags (FIGS. 26 and 27).Although a significant overlap of the charge variant species of the twomAbs were observed, these peaks species were not impacted from theincrease in the mAb content of the IV infusion bag. Pertuzumab alone orTrastuzumab alone samples in PO or PVC IV infusion bags showed nochanges before and after exposure to 5° C. or 30° C. These results areconsistent with the 840 mg dose I study.

Conclusion

All physicochemical assays indicate no significant changes in themixtures (up to 840 mg Pertuzumab and 720 mg Trastuzumab for a 1560 mgtotal dose) or in the individual Pertuzumab (up to 840 mg) andTrastuzumab (up to 720 mg) IV infusion bags (PO or PVC) for T0 to T24hours at 5° C. or 30° C. Furthermore, the potency of the mixture (up to840 mg) and the individual mAbs before and after storage werecomparable. No differences were observed in the IV bags that containedthe admixture of Pertuzumab and Trastuzumab when compared to theindividual mAb components in IV bags over the course of this study. Thecurrent study also demonstrates that many of the assays used to measurethe individual mAbs were sufficient to qualitatively characterize theadmixture.

Example 7 Co-Administration of Pertuzumab and Trastuzumab, andCombination Therapy with Vinorelbine

This is a randomized, two-arm, open-label, multicenter Phase II trial toevaluate Pertuzumab in patients with HER2-positive advanced breastcancer (metastatic or locally advanced) who have not previously receivedsystemic non-hormonal anticancer therapy in the metastatic setting. Thestudy design is shown in FIG. 28.

Patients are randomly assigned in a 2:1 ratio to one of two treatmentarms:

Pertuzumab given in combination with Trastuzumab and vinorelbine (Arm A)

Trastuzumab and vinorelbine (control arm Arm B)

Arm A will consist of two cohorts as follows:

Cohort 1: (first 95 patients): Pertuzumab and Trastuzumab administeredsequentially in separate infusion bags, followed by vinorelbine.Patients will receive Pertuzumab followed by Trastuzumab sequentially inseparate infusion bags, followed by vinorelbine.

Pertuzumab (IV Infusion)

Administered on Day 1 of the first treatment cycle as a loading dose of840 mg, followed by 420 mg on Day 1 of each subsequent 3 weekly cycle.

Initial infusions of Pertuzumab will be administered over 90 (±10)minutes and patients observed for at least 30 minutes from the end ofinfusion for infusion-related symptoms such as fever, chills etc.Interruption or slowing of the infusion may reduce such symptoms. If theinfusion is well tolerated, subsequent infusions may be administeredover 30 (±10) minutes with patients observed for a further 30 minutes.

Trastuzumab (IV Infusion)

Day 1 of the first treatment cycle as a loading dose of 8 mg/kg,followed by 6 mg/kg on Day 1 of each subsequent 3 weekly cycle; to beadministered in line with product labeling.

Vinorelbine (IV Infusion after Trastuzumab)

Day 1 and Day 8 of the first treatment cycle at a dose of 25 mg/m²followed by 30-35 mg/m² on Day 1 and Day 8 of each subsequent 3 weeklycycle; to be administered in line with product labeling.

Cohort 2: The second 95 patients will receive Pertuzumab and Trastuzumabadministered together in a single infusion bag from Cycle 2 onwards,followed by vinorelbine.

Cycle 1 Dosing

For the first cycle of treatment, Pertuzumab and Trastuzumab will beadministered in separate infusion bags as described for Cohort 1.

Vinorelbine will be administered after Pertuzumab and Trastuzumab asdescribed for Cohort 1.

Subsequent Cycle Dosing

If administration of all three drugs was well tolerated in Cycle 1, thenon Day 1 of each subsequent 3 weekly treatment cycle. Pertuzumab 420 mgand Trastuzumab 6 mg/kg will be given together in a single infusion bag.

The first combined infusion of Pertuzumab and Trastuzumab should beadministered over 90 (±10) minutes with cardiac monitoring and closeobservation for infusion-associated reactions during the procedure,followed by a 60 minute observation period. If this first combinedinfusion is well tolerated, subsequent combined infusions can beadministered over 60 (±10) minutes followed by a 30 minute observationperiod with cardiac monitoring.

Vinorelbine will be administered after Pertuzumab and Trastuzumab asdescribed for Cohort 1.

Control arm—Arm B

A total of 95 patients will be randomized to arm B.

Trastuzumab (IV Infusion)

Day 1 of the first treatment cycle as a loading dose of 8 mg/kg,followed by 6 mg/kg on Day 1 of each subsequent 3 weekly cycle; to beadministered in line with product labeling.

Vinorelbine (IV Infusion after Trastuzumab)

Day 1 and Day 8 of the first treatment cycle at a dose of 25 mg/m²followed by 30-35 mg/m² on Day 1 and Day 8 of each subsequent 3 weeklycycle; to be administered in line with product labeling.

Efficacy Outcomes:

Primary

-   -   To compare objective overall response rates (ORR) assessed by a        blinded independent review committee (IRC) of Pertuzumab given        in combination with Trastuzumab and vinorelbine versus        Trastuzumab and vinorelbine

Secondary

-   -   Within the Pertuzumab treatment group to compare the efficacy        and safety of Pertuzumab and Trastuzumab administered together        in a single infusion bag versus conventional sequential        administration in separate infusion bags    -   To compare Pertuzumab given in combination with Trastuzumab and        vinorelbine versus Trastuzumab and vinorelbine with respect to:        -   ORR assessed by the Investigator        -   Time to response assessed by IRC and Investigator        -   Duration of response assessed by IRC and Investigator        -   Progression free survival (PFS)        -   Time to progression (TTP)        -   Overall survival (OS)        -   Safety and tolerability

Quality of life (EQ-SD and FACT-B questionnaires)

Inclusion Criteria

Patients must meet the following criteria to be eligible for this studyaccording to the timing of the Schedule of Assessments:

-   -   1. Female or male patients aged 18 years or older    -   2. Histologically or cytologically confirmed and documented        adenocarcinoma of the breast with metastatic or locally advanced        disease not amenable to curative resection    -   3. HER2-positive (defined as either imnnunohistochemistry (IHC)        3+ or in situ hybridization (ISH) positive) as assessed by local        laboratory on primary or metastatic tumor (ISH positivity is        defined as a ratio of 2.0 or greater for the number of HER2 gene        copies to the number of signals for CEP17, or for single probe        tests, a HER2 gene count greater than 4).    -   4. At least one measurable lesion and/or non-measurable disease        evaluable according to Response Evaluation Criteria In Solid        Tumors (RECIST) version 1.1    -   5. ECOG performance status 0 or 1    -   6. Left ventricular ejection fraction (LVEF) of at least 50%    -   7. Negative pregnancy test in women of childbearing potential        (premenopausal or less than 12 months of amenorrhea        post-menopause, and who have not undergone surgical        sterilization)    -   8. For women of childbearing potential who are sexually active,        agreement to use a highly-effective, non-hormonal form of        contraception or two effective forms of non-hormonal        contraception during and for at least 6 months post study        treatment    -   9. Fertile males willing and able to use effective non-hormonal        means of contraception (barrier method of contraception in        conjunction with spermicidal jelly, or surgical sterilization)        during and for at least 6 months post-study treatment    -   10. Life expectancy of at least 12 weeks

Exclusion Criteria

Patients who meet any of the following exclusion criteria will not beeligible for this study:

-   1. Previous systemic non-hormonal anticancer therapy in the    metastatic or locally advanced breast cancer setting-   2. Previous approved or investigative anti-HER2 agents in any breast    cancer treatment setting, except Trastuzumab in the adjuvant or    neoadjuvant setting-   3. Disease progression while receiving Trastuzumab in the adjuvant    or neoadjuvant setting-   4. Disease-free interval from completion of adjuvant or neo-adjuvant    systemic non-hormonal treatment to recurrent disease of less than 6    months-   5. History of persistent grade 2 or higher (NCI-CTC, Version 4.0)    hematological toxicity resulting from previous adjuvant or    neoadjuvant therapy-   6. Radiographic evidence of central nervous system (CNS) metastases    as assessed by CT or MRI-   7. Current peripheral neuropathy of grade 3 or greater (NCI-CTC,    Version 4.0)-   8. History of other malignancy within the last 5 years, except for    carcinoma in situ of the cervix or basal cell carcinoma-   9. Serious uncontrolled concomitant disease that would    contraindicate the use of any of the investigational drugs used in    this study or that would put the patient at high risk for treatment    related complications-   10. Inadequate organ function, evidenced by the following laboratory    results:    -   Absolute neutrophil count <1,500 cells/mm³    -   Platelet count <100,000 cells/mm³    -   Hemoglobin <9 g/dL    -   Total bilirubin greater than upper limit of normal (ULN) (unless        the patient has documented Gilbert's syndrome)    -   AST (SGOT) or ALT (SGPT)>2.5×ULN    -   AST (SGOT) or ALT (SGPT)>1.5×ULN with concurrent serum alkaline        phosphatase >2.5×ULN; Serum alkaline phosphatase may be >2.5×ULN        only if bone metastases are present and AST (SGOT) and ALT        (SGPT)<1.5×ULN    -   Serum creatinine >2.0 mg/dL or 177 μmol/L    -   International normalized ratio (INR) and activated partial        thromboplastin time or partial thromboplastin time (aPTT or        PTT)>1.5×ULN (unless on therapeutic coagulation)-   11. Uncontrolled hypertension (systolic >150 mm Hg and/or    diastolic >100 mm Hg) or clinically significant (i.e. active)    cardiovascular disease: cerebrovascular accident (CVA)/stroke or    myocardial infarction within 6 months prior to first study    medication, unstable angina, congestive heart failure (CHF) of New    York Heart Association (NYHA) grade II or higher, or serious cardiac    arrhythmia requiring medication-   12. Current known infection with HIV, HBV, or HCV-   13. Dyspnea at rest due to complications of advanced malignancy, or    other disease requiring continuous oxygen therapy-   14. Major surgical procedure or significant traumatic injury within    28 days prior to randomization or anticipation of need for major    surgery during the course of study treatment-   15. Receipt of intravenous (TV) antibiotics for infection within 14    days prior to randomization-   16. Current chronic daily treatment with corticosteroids (dose    equivalent to or greater than 10 mg/day methylprednisolone),    excluding inhaled steroids-   17. Known hypersensitivity to any of the study medications or to    excipients of recombinant human or humanized antibodies-   18. History of receiving any investigational treatment within 28    days prior to randomization-   19. Concurrent participation in any clinical trial

It is anticipated that the treatment herein will demonstrate the safetyand efficacy of co-administration of pertuzumab and Trastuzumab from thesame intravenous (IV) bag to patients with HER2-positive cancer(exemplified by HER2-positive breast cancer), as well as the safety andefficacy of Pertuzumab in combination in vinorelbine according to anyone or more of the primary or secondary efficacy outcomes above.

Example 8 Pertuzumab Combined with Aromatase Inhibitors

This example is a randomized, two-arm, open-label, multicenter phase IIstudy demonstrating the efficacy and safety of Pertuzumab given incombination with Trastuzumab plus an aromatase inhibitor in first linepatients with HER2-positive and hormone receptor-positive advanced(metastatic or locally advanced) breast cancer. The study design isshown in FIG. 29.

Primary Objectives

To compare progression-free survival (PFS) of Pertuzumab given incombination with Trastuzumab plus an aromatase inhibitor (AI) versusTrastuzumab plus an AI.

Secondary Objectives

To compare Pertuzumab given in combination with Trastuzumab plus an AIversus Trastuzumab plus an AI with respect to:

Overall survival (OS)

Overall response rate (ORR)

Clinical benefit rate (CBR)

Duration of response

Time to response

Safety and tolerability

Quality of life (EQ-5D questionnaires)

Trial Design

Patients will be randomly assigned in a 1:1 ratio to one of twotreatment arms:

Pertuzumab in combination with Trastuzumab plus an AI (Arm A).

Trastuzumab plus an AI (control arm Arm B).

At the investigator's discretion, patients may also receive inductionchemotherapy (a taxane, either Docetaxel or paclitaxel), in combinationwith the assigned monoclonal antibody treatment arm up to the first 18weeks of the treatment period. In patients receiving inductionchemotherapy, treatment with the AI will start after the chemotherapyinduction phase.

Stratification factors for analysis will be:

-   -   Chosen to receive induction chemotherapy (Yes/No).    -   Time since adjuvant hormone therapy (<12 months, ≥12 months, or        no prior hormone therapy).

Patients with HER2-positive and hormone receptor-positive (estrogenreceptor (ER)-positive and/or progesterone receptor (PgR)-positive)advanced breast cancer (metastatic or locally advanced) who have notpreviously received systemic nonhormonal anticancer therapy in themetastatic setting.

Inclusion Criteria

1. Age greater than or equal to 18 years.2. Postmenopausal status >1 year (fulfilling one or more of NationalComprehensive Cancer Network (NCCN) guideline criteria, Version 2.2011).3. Histologically or cytologically confirmed and documentedadenocarcinoma of the breast with metastatic or locally advanced diseasenot amenable to curative resection.4. HER2-positive (defined as either IHC 3+ or ISH positive) as assessedby local laboratory on primary or metastatic tumor (ISH positivity isdefined as a ratio of 2.0 or greater for the number of HER2 gene copiesto the number of signals for CEP17, or for single probe tests, a HER2gene count greater than 4).5. Hormone receptor-positive defined as ER-positive and/or PgR-positiveassessed locally as defined by institutional criteria.6. At least one measurable lesion and/or non-measurable diseaseevaluable according to Response Evaluation Criteria In Solid Tumors(RECIST) version 1.1.7. ECOG performance status 0 or 1.8. Left ventricular ejection fraction (LVEF) of at least 50%.9. Life expectancy of at least 12 weeks.

Exclusion Criteria

1. Previous systemic non-hormonal anticancer therapy in the metastaticor locally advanced breast cancer setting.2. Disease-free interval from completion of adjuvant or neo-adjuvantsystemic non-hormonal treatment to recurrence of within 6 months.3. Previous approved or investigative anti-HER2 agents in any breastcancer treatment setting, except Trastuzumab and/or lapatinib in theneoadjuvant or adjuvant setting.4. Disease progression while receiving Trastuzumab and/or lapatinib inthe adjuvant setting.5. History of persistent grade 2 or higher (NCI-CTC. Version 4.0)hematological toxicity resulting from previous adjuvant or neo-adjuvanttherapy.6. Radiographic evidence of central nervous system (CNS) metastases asassessed by CT or MRI.7. Current peripheral neuropathy of grade 3 or higher (NCI-CTC, Version4.0).8. History of other malignancy within the last 5 years, except forcarcinoma in situ of the cervix or basal cell carcinoma.9. Serious uncontrolled concomitant disease that would contraindicatethe use of any of the investigational drugs used in this study or thatwould put the patient at high risk for treatment related complications.10. Inadequate organ function, evidenced by the following laboratoryresults:

Absolute neutrophil count <1,500 cells/mm₃.

Platelet count <100,000 cells/mm₃.

Hemoglobin <9 g/dL.

Total bilirubin greater than the upper limit of normal (ULN) (unless thepatient has documented Gilbert's syndrome).

AST (SGOT) or ALT (SGPT)>2.5×ULN.

AST (SGOT) or ALT (SGPT)>1.5×ULN with concurrent serum alkalinephosphatase>2.5×ULN Serum alkaline phosphatase may be >2.5×ULN only ifbone metastases are present and AST (SGOT) and ALT (SGPT)<1.5×ULN.

Serum creatinine >2.0 mg/dL or 177 μmoL/L.

International normalized ratio (INR) and activated partialthromboplastin time (aPTT) or partial thromboplastin time (PTT)>1.5×ULN(unless on therapeutic coagulation).

11. Uncontrolled hypertension (systolic >150 mm Hg and/or diastolic >100mm Hg) or clinically significant (i.e. active) cardiovascular disease:cerebrovascular accident (CVA)/stroke or myocardial infarction within 6months prior to first study medication, unstable angina, congestiveheart failure (CHF) of New York Heart Association (NYHA) grade II orhigher, or serious cardiac arrhythmia requiring medication.12. Current known infection with HIV, HBV, or HCV.13. Dyspnea at rest due to complications of advanced malignancy, orother disease requiring continuous oxygen therapy.14. Major surgical procedure or significant traumatic injury within 28days prior to randomization or anticipation of needed for major surgeryduring the course of study treatment.15. Lack of physical integrity of the upper gastrointestinal tract,clinically significant malabsorption syndrome, or inability to take oralmedication.16. Receipt of intravenous antibiotics for infection within 14 daysprior to randomization.17. Current chronic daily treatment with corticosteroids (dose of 10mg/day methylprednisolone equivalent), excluding inhaled steroids.18. Known hypersensitivity to any of the study medications or toexcipients of recombinant human or humanized antibodies.19. History of receiving any investigational treatment within 28 daysprior to randomization.20. Concurrent participation in any clinical trial.

Arm A

Pertuzumab (IV Infusion)

Administered on Day 1 of the first treatment cycle as a loading dose of840 mg, followed by 420 mg on Day 1 of each subsequent 3 weekly cycle.

Initial infusions of Pertuzumab will be administered over 90 (±10)minutes and patients observed for at least 30 minutes from the end ofinfusion for infusion-related symptoms such as fever, chills etc.Interruption or slowing of the infusion may reduce such symptoms. If theinfusion is well tolerated, subsequent infusions may be administeredover 30 (±10) minutes with patients observed for a further 30 minutes.

Trastuzumab (IV infusion administered after Pertuzumab) Day 1 of thefirst treatment cycle as a loading dose of 8 mg/kg, followed by 6 mg/kgon Day 1 of each subsequent 3 weekly cycle; to be administered in linewith product labeling.

AI (Oral)

Administered in line with product labeling (anastrozole: 1 mg oncedaily; letrozole: 2.5 mg once daily).

Induction Chemotherapy

Patients receiving induction chemotherapy up to the first 18 weeks ofthe treatment period will receive a taxane (Docetaxel every 3 weeks orpaclitaxel weekly), administered in line with the respective productlabeling. Chemotherapy will be administered after the monoclonalantibody (Pertuzumab and/or Trastuzumab) infusions.

In patients receiving induction chemotherapy treatment with the AI willstart after the chemotherapy induction phase.

Control Arm—Arm B

Trastuzumab (IV Infusion)

Day 1 of the first treatment cycle as a loading dose of 8 mg/kg,followed by 6 mg/kg on Day 1 of each subsequent 3 weekly cycle; to beadministered in line with product labeling.

AI (Oral)

Administered in line with product labeling (anastrozole: 1 mg oncedaily; letrozole: 2.5 mg once daily).

Induction Chemotherapy

Same as for investigational arm.

Primary Efficacy Outcome

PFS (defined as the time from randomization until the firstradiographically documented

Progression of Disease or Death from any Cause, Whichever Occurs First).

Secondary Efficacy Outcome

OS

ORR

CBR

Duration of response

Time to response

Safety

Incidence and severity of adverse events (AEs) and serious adverseevents (SAEs)

Incidence of CHF

LVEF over the course of the study

Laboratory test abnormalities

It is anticipated that the combination of Pertuzumab. Trastuzumab and AIwill be safe and effective in the patient population and that theaddition of Pertuzumab to Trastuzumab and an AI will extendprogression-free survival (PFS) compared to Trastuzumab plus an AIwithout Pertuzumab.

Example 9 Pertuzumab for Improving Overall Survival (OS) in CancerPatients

Background:

In the CLEOPATRA study in Example 3 above, 808 patients withHER2-positive first-line (1 L) metastatic breast cancer (MBC) wererandomized to treatment with Placebo+Trastuzumab+Docetaxel (Pla+T+D) orPertuzumab+Trastuzumab+Docetaxel (P+T+D). The primary endpoint ofindependently reviewed progression-free survival was significantlyimproved with P+T+D vs Pla+T+D (hazard ratio (HR)=0.62; P<0.0001;medians. 18.5 vs 12.4 mths) (Example 3 above). This example includes asecond interim overall survival (OS) analysis after longer follow-up.

Methods:

This interim overall survival (OS) analysis was performed applying theLan-DeMets α-spending function with the O'Brien-Fleming (OBF) stoppingboundary to maintain the overall Type I error at 5%. Based on the numberof OS events observed, the OBF boundary for statistical significance atthis analysis was P≤0.0138. The log-rank test, stratified by priortreatment status and geographic region, was used to compare OS betweenarms in the intention-to-treat population. The Kaplan-Meier approach wasused to estimate the median OS in both arms; a stratified Coxproportional hazard model was used to estimate HR and 95% CIs. Subgroupanalyses of OS were performed for the stratification factors and otherkey baseline characteristics.

Results:

At the time of this analysis, median follow-up was 30 months and 267deaths (69% of planned events for the final analysis) had occurred. Theresults showed a statistically significant improvement in OS in favor ofP+T+D (HR=0.66; 95% confidence interval (CI), 0.52-0.84; P=0.0008). ThisHR represents a 34% reduction in the risk of death. The analysisachieved statistical significance and is therefore considered theconfirmatory OS analysis. The median OS was 37.6 mths in the Pla arm andhas not yet been reached in the P arm. The treatment effect wasgenerally consistent in predefined subgroups based on baseline variablesand stratification factors, including: prior (neo)adjuvant therapy(HR=0.66; 95% CI, 0.46-0.94); no prior (neo)adjuvant therapy (HR=0.66;95% CI, 0.47-0.93); prior (neo)adjuvant T (HR=0.68; 95% CI, 0.30-1.55);hormone receptor-negative disease (HR=0.57; 95% CI, 0.41-0.79); andhormone receptor-positive disease (HR=0.73; 95% CI, 0.50-1.06).Kaplan-Meier estimates of OS rates show survival benefit with P+T+D at1, 2, and 3 yrs.

TABLE 14 Overall Survival Benefit with Pertuzumab Pla + T + D P + T + DΔ Survival rates, % 1 yr  89.0 94.4 5.4 2 yrs 69.4 80.7 11.3 3 yrs 50.465.8 15.4

The majority of pts received anti-cancer therapy after discontinuationof study treatment (64% Pla arm. 56% P arm). Subsequent therapy withHER2-directed agents (T, lapatinib, T emtansine) was balanced betweenarms. Causes of death remained unchanged from the first interim OSanalysis, with the most common cause being progressive disease. Adverseevents leading to death were rare and balanced between arms.

Conclusions:

Treatment of patients with HER2-positive 1 L MBC with P+T+D comparedwith Pla+T+D was associated with an improvement in OS, which was bothstatistically significant and clinically meaningful. These results showthat combined HER2 blockade and chemotherapy using the P+T+D regimen canbe considered a standard of care for patients with HER2-positive MBC inthe 1L setting.

These data regarding OS can be included on the package insert withprescribing information regarding Pertuzumab in an article ofmanufacture as in Example 4 above, for example.

Example 10 Pertuzumab and Trastuzumab with a Taxane as First-LineTherapy for Patients with HER2-Positive Advanced Breast Cancer (PERUSE)

Background:

Pertuzumab (P), a humanized monoclonal antibody, inhibits signalingdownstream of HER2 by binding to the dimerization domain of the receptorand preventing heterodimerization with other HER family members. Theepitope recognized by P is distinct from that bound by Trastuzumab (H)and so their complementary mechanisms of action result in a morecomprehensive HER2 blockade. Data from the phase III trial CLEOPATRAshowed significantly improved PFS in patients (pts) receivingP+H+docetaxel compared with H+docetaxel+placebo as first-line treatmentfor HER2-positive metastatic breast cancer (BC).

Trial Design:

This is a phase IIIb, multicenter, open-label, single-arm study in ptswith HER2-positive metastatic or locally recurrent BC who have not beentreated with systemic nonhormonal anticancer therapy for metastaticcancer. Pts will receive, P: 840 mg initial dose, 420 mg q3w IV; H: 8mg/kg initial dose, 6 mg/kg q3w IV; taxane: docetaxel, paclitaxel, ornab-paclitaxel according to local guidelines. Treatment will beadministered until disease progression or unacceptable toxicity. Aplanned protocol amendment will allow hormone receptor-positive pts toreceive endocrine therapy alongside P+H after completion of taxanetherapy, in line with clinical practice.

Eligibility Criteria:

At baseline, pts must have an LVEF of ≥50%, an ECOG PS of 0, 1, or 2, adisease-free interval of ≥6 months, and must not have received prioranti-HER2 agents for the treatment of metastatic BC. Prior H and/orlapatinib in the (neo)adjuvant setting is permitted, providing there wasno disease progression during treatment. Pts must not have experiencedother malignancies within the last 5 yrs other than carcinoma in situ ofthe cervix or basal cell carcinoma. There must be no clinical orradiographic evidence of CNS metastases or clinically significantcardiovascular disease.

Specific Aims:

As H was not widely available in the (neo)adjuvant setting prior toCLEOPATRA recruitment, a relatively low proportion of pts in CLEOPATRAhad previously received H. PERUSE will assess the safety andtolerability of P+H+choice of taxane as first-line therapy for pts withHER2-positive metastatic or locally advanced BC in a pt populationlikely to have experienced wider exposure to prior H therapy.

Statistical Methods:

The primary endpoints of the PERUSE study are safety and tolerability.Secondary endpoints include PFS, OS. ORR. CBR, duration of response,time to response and QoL. The final analysis will be performed when 1500pts have been followed up for at least 12 months after the last ptreceives last study treatment unless they have been lost to follow-up,withdrawn consent, or died, or if the study is prematurely terminated bythe sponsor. Safety analyses are planned after enrollment of ˜350, 700,and 1000 pts. Additionally, a data and safety monitoring board willreview safety data after ˜0.50 pts have been enrolled and then every 6months.

It is anticipated that the Pertuzumab and Trastuzumab with a taxane willbe effective as first-line therapy for patients with HER2-positiveadvanced breast cancer according to the protocol in this example.

Example 11 Pertuzumab in Combination with Chemotherapy in Low HER3Ovarian Cancer

Epithelial ovarian cancer, along with primary peritoneal, and fallopiantube carcinoma, is the fifth leading cause of cancer-related deaths inwomen in Europe (Bray et al. Int. J. Cancer 113:977-90 (2005)). Ovariancancer is often not diagnosed until it has progressed to an advancedstage, at which point the standard treatment is surgical resectionfollowed by chemotherapy. Although the addition of taxanes toplatinum-based chemotherapy has resulted in approximately 80% ofpatients achieving complete response (CR), the disease recurs in mostpatients, and more than 50% of patients diagnosed with epithelialovarian cancer eventually die from their disease (Du Bois et al. Cancer115:1234-1244 (2009)). Following failure of platinum-based chemotherapy,there are few therapeutic options. Patients with platinum-sensitivedisease (disease recurrence occurs more than 6 months after last cycleof platinum-based chemotherapy) are often retreated with platinum-basedtherapy and have a progression-free survival (PFS) of approximately9-10; however, for patients with primary platinum-resistant disease, theprognosis is considerably worse. For these patients, re-treatment withplatinum-based therapy or surgery is not reasonable, instead, patientswith platinum-resistant are often treated with single-agent chemotherapysuch as topotecan, pegylated liposomal doxorubicin (PLD), paclitaxel andgemcitabine.

Objective response rates for patients with platinum-resistant diseaseranges between 10-20% while median progression free survival (PFS)ranges between 3.5-4 months. Platinum-resistant disease is not curable;the goals of treatment for these patients include palliation ofsymptoms, prolonged survival and improvements in quality of life (QoL).Overall, results from major clinical trials conducted over the last 20years show that the median PFS for patients with advanced disease rangesbetween 16-23 months while median overall survival (OS) ranges between31-65 months.

The majority of ovarian cancer cell lines and many ovarian cancer biopsysamples express all members of the HER family of receptors (Campiglio etal. J. Cell Biochem 73:522-32 (1999)). EGFR and HER2 have been studiedthe most extensively, and multiple agents targeting the receptor orassociated intracellular tyrosine kinases have been tested.

In a recent study, quantitative HER2 protein analyses demonstrated thatmalignant ovarian tumors have significantly higher levels of HER2compared with benign ovarian tumors and normal ovaries. Furthermore, acorrelation between HER2 and HER3 protein levels has been seen(Steffensen et al. Int J Oncol. 33:195-204 (2008)). Studies in cellculture systems have shown that heregulin-activated HER3-HER2heterodimers elicit the strongest proliferative and transformationresponses of any possible receptor combination (Pinkas-Kramarski et al.EMBO J. 15:2452-67 (1996); Riese et al. Mol Cell Biol 15:5770-6 (1995).Erratum in: Mol Cell Biol 16:735 (1996)). The potency of these biologicresponses is likely the result of the dual and efficient activation ofthe MAP kinase and PI3 kinase pathways. Furthermore, HER3 is the mostpotent activator of the PI3 kinase/AKT pathway (Olayioye et al. EMBO J19:3159-67 (2000)). Studies in HER2-amplified breast cancer cell linesshow that HER3 but not EGFR was critical for HER2 signaling, and thatHER3 inhibited growth in three-dimensional culture and induced rapidtumor regression of in vivo xenografts (Lee-Hoeflich et al. Cancer Res68:5878-87 (2008)).

Additionally, HER3 expression has been implicated as a possible riskfactor in ovarian cancer (Tanner et al. J Clin Oncol 24:4317-23 (2006)).

In a Phase II multicenter trial (TOC2689g) in patients with advancedovarian cancer that recurred after treatment with or were refractory toplatinum-based chemotherapy, patients who were enrolled in Cohort 1(n=61) received a loading dose of 840 mg Pertuzumab, followed by 420 mgPertuzumab on Day 1 of each 3-week cycle, and patients in Cohort 2(n=62) received 1050 mg Pertuzumab on Day 1 of each 3-week cycle.Similar outcomes were observed in both cohorts in terms of overallresponse rate and median PFS. Eight patients (4 in each cohort) hadevidence of stable disease (SD) lasting at least 6 months. Median PFSand OS were 6.6 weeks and 52.7 weeks, respectively, for the overallpopulation.

The results of this study led to two randomized Phase II trials inplatinum-sensitive and platinum-resistant populations. In StudyTOC3258g, the efficacy and safety of gemcitabine+Pertuzumab versusgemcitabine+placebo were evaluated in patients with advanced ovarian,primary peritoneal, or fallopian tube cancer that was resistant toplatinum-based chemotherapy (Amler et al. J Clin Oncol 26:5552 (2008)).The study allowed patients to cross over to receive Pertuzumab at thetime of disease progression. There was a median PFS of 2.6 months in thegemcitabine+placebo arm and 2.9 months in the gemcitabine+Pertuzumabarm. Median OS was similar between the treatment arms. Of the mostcommon adverse events (AEs), those increased (by at least 6 patients) inthe Pertuzumab-treated cohort included fatigue, nausea, diarrhea, backpain, dyspepsia, stomatitis, headache, epistaxis, rhinorrhea, rash, andGrade 3-4 neutropenia.

In Study BO17931, 149 patients with ovarian cancer who experienced arecurrence 6 months after a platinum-based therapy were randomized toreceive a combination of paclitaxel and carboplatin or gemcitabine withor without Pertuzumab. After 6 treatment cycles, chemotherapy wasdiscontinued, and patients in the chemotherapy+Pertuzumab arm continuedto receive Pertuzumab alone for up to 11 additional cycles (total of 17cycles of Pertuzumab). There were no significant differences in the PFSor OS for the overall group. Median PFS was 34.1 weeks for thechemotherapy+Pertuzumab group versus 31.3 for the chemotherapy alonegroup; however, an exploratory subset analyses of HER3 mRNA expressionwith a treatment-free interval of 6-12 months indicated a trend towardclinical benefit in patients who express high levels of HER3 mRNA (Kayeet al. J Clin Oncol 26:5520 (2008)).

Archival tissue samples from patients enrolled in both randomized PhaseII studies were examined by quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) for mRNA expression levels of theHER receptors EGFR, HER2, HER3, and two HER ligands: amphiregulin andbetacellulin.

Only tumor HER3 mRNA expression was associated with a significantdifference in PFS. For patients who achieved a clinical response. PRswere observed in 9 patients on the gemcitabine+Pertuzumab arm and 3 onthe gemcitabine+placebo arm. Six of the gemcitabine+Pertuzumab patientswith PRs had tumor HER3 mRNA levels lower than the median level. Incontrast, no patients in the gemcitabine+placebo arm whose tumor HER3mRNA levels were lower than the median level of the study populationexperienced a PR. An additional 6 patients achieved PRs. and all ofthese patients had tumor HER3 mRNA levels at or above the median levelof the study population. Of these patients, 3 receivedgemcitabine+Pertuzumab and 3 received gemcitabine+placebo, suggesting noeffect of Pertuzumab in this population.

Patients with low HER3 mRNA expression (lower than the median level ofthe study population) demonstrated a PFS hazard ratio (HR) of 0.32 incontrast to 1.68 for patients with HER3 mRNA expression greater than orequal to the median level; i.e. the effect of adding Pertuzumab trendedin the opposite direction. No significant benefit was detected in OS forpatients with low HER3 mRNA expression; however, a trend toward greaterOS was observed in patients receiving Pertuzumab. The OS for patientsexpressing high HER3 mRNA expression demonstrated an HR of 1.59.

To assess the prognostic value. HER3 mRNA expression was correlated withPFS and OS for patients in the gemcitabine+placebo arm. Median PFS was1.4 months for patients with low HER3 mRNA expression (n=35), comparedwith 5.5 months for patients with high HER3 mRNA expression (n=24).Similarly, median OS for patients with low HER3 mRNA expression was 8.4months, compared with 18.2 months for patients with high HER3 mRNAexpression.

In Study BO17931, in patients with low HER3 mRNA expression (lower thanthe median level of this study population), no treatment effect wasseen. However, in an exploratory analysis of patients with atreatment-free interval of 6-12 months, there was a trend toward benefitfor the combination of chemotherapy with Pertuzumab in terms of PFS.

Overview of this Study

This is a multicenter trial with two parts; a non-randomized safetyrun-in Part 1 and a randomized, double-blind Part 2.

Part 1 will be performed to assess safety and tolerability of Pertuzumabin a new combination with two chemotherapeutic agents (topotecan orpaclitaxel). Part 2 of the trial is a randomized, double-blind, placebocontrolled, two-arm, multicenter, prospective trial of Pertuzumab incombination with chemotherapy (topotecan, paclitaxel, or gemcitabine).Patients will receive trial medication until disease progression as perthe Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1,disease progression according to the Gynecologic Cancer Intergroup(GCIG) criteria of CA-125 assessable disease, unacceptable toxicity,withdrawal of consent, or death. PFS will be assessed in Part 1 of thetrial, but due to small number of patients and PFS events per cohort,results will be descriptive only. The trial design for Part 1 of thestudy is provided in FIG. 30.

In Part 2 of the trial, patients will be randomized in a 1:1 ratio toreceive either:

Arm A: Pertuzumab in combination with chemotherapy (topotecan,paclitaxel, or gemcitabine), or

Arm B: Pertuzumab-placebo plus chemotherapy (topotecan, paclitaxel, orgemcitabine).

The allocation of study medication will be double-blind with respect towhether the patient receives Pertuzumab or Pertuzumab-placebo. Thechemotherapy agent allocated will be at the discretion of theinvestigator.

Stratification factors for Part 2 of the trial will be:

-   -   Selected chemotherapy cohort (topotecan vs. paclitaxel vs.        gemcitabine).    -   Previous anti-angiogenic therapy (yes vs. no). If a patient has        previously participated in a blinded trial with an        anti-angiogenic agent, the patient will be enrolled in the same        stratum with patients known to have previously received an        anti-angiogenic agent.    -   Treatment-free interval (TFI) since platinum therapy (strictly        less than 3 months vs. 3 to 6 months inclusive, prior to first        study treatment).

The trial design for Part 2 of the study is provided in FIG. 31.

Primary Objectives of the Study:

Part 1: The primary objective for Part 1 of this study is to determinethe safety and tolerability of Pertuzumab in combination with eithertopotecan or paclitaxel.

Part 2: The primary objective for Part 2 of this study is to determineif Pertuzumab plus chemotherapy is superior to placebo plus chemotherapyas measured by PFS.

Secondary Objectives of the Study:

Part 1: The secondary objective for Part 1 of this study is to evaluatedescriptively the PFS of Pertuzumab in combination with either topotecanor paclitaxel.

Part 2: The secondary objectives for Part 2 of this study are todetermine if Pertuzumab plus chemotherapy is superior to placebo pluschemotherapy with respect to:

-   -   OS.    -   Objective response rate.    -   Biological progression-free interval (PFI_(BIO)).    -   Safety and tolerability.    -   QoL.

Efficacy Outcome Measures

The following efficacy outcome measures will be measured in Part 1 ofthe trial:

-   -   PFS, which is defined as the time from randomization into Part 1        of the trial until disease progression per RECIST version 1.1 or        according to GCIG criteria in CA-125 assessable disease or death        from any cause, whichever occurs first.

The efficacy outcome measures for Part 2 of the trial are as follows:

-   -   PFS, which is defined as the time from randomization into Part 2        of the trial, until disease progression per RECIST version 1.1        or according to GCIG criteria in CA-125 assessable disease or        death from any cause, whichever occurs first.    -   OS, defined as the time from randomization into Part 2 of the        trial until death from any cause.    -   Objective response rate (ORR), which will be based on RECIST        version 1.1 and assessed by the best (confirmed) overall        response (BOR); defined as the best response recorded from the        start of the treatment in Part 2 of the trial until disease        progression/recurrence (taking as reference for PD, the smallest        measurements recorded since the treatment started in Part 2 of        the trial). Patients need to have two consecutive assessments of        partial response (PR) or complete response (CR) to be a        responder. PR or CR has to be confirmed by 2 consecutive tumor        evaluations spaced at least 4 weeks apart. Only patients with        measurable disease at baseline will be included in the analysis        of objective response.    -   Patients who have a response as per RECIST version 1.1 and using        the 50% response criteria for CA-125 are defined as responders,        whereas patients who only have response as defined per RECIST        are defined as RECIST responders. Patients who do not have a        response as per RECIST, but have a response defined using the        50% response criteria for CA-125 are defined as CA-125        responders.    -   PFI_(BIO), defined on the basis of a progressive serial        elevation of serum CA-125 (assessed according to the CGIG        criteria) as the time from the date of randomization into Part 2        of the trial to first documented increase in CA-125 levels to:        two times the upper limit of normal (for patients with normal        pretreatment CA-125 or elevated pretreatment CA-125 and initial        normalization on-treatment), or two times the nadir value (for        patients with elevated baseline CA-125 that did not normalize        on-treatment).

Safety Outcome Measures

In Part 1 of the study safety and tolerability will be assessed afterall patients have received 3 cycles of treatment.

In addition, safety outcome measures for this study will be assessed inboth Parts 1 and 2 of the study, and are as follows:

-   -   Incidence, nature, and severity of all AEs, serious adverse        events (SAEs). AEs with NCI-CTCAE version 4.0 Grades ≥3, and AEs        that caused premature withdrawal from study medication.    -   Premature withdrawal from the study and study treatment.    -   Cardiac disorders/Incidence of congestive heart failure    -   Laboratory test abnormalities.    -   Left ventricular ejection fraction

Inclusion Criteria

1. Female patients aged 18 years or older.

2. Low HER3 mRNA expression levels (concentration ratio equal or lowerthan 2.81, as assessed by qRT-PCR on a COBAS z480® (instrument).

3. Histologically or cytologically confirmed and documented epithelialovarian cancer that is platinum-resistant or refractory (defined asprogression within 6 months from completion of a minimum of 4 platinumtherapy cycles or progression during platinum therapy).

4. At least one measurable lesion and/or non-measurable diseaseaccording to RECIST version 1.1, or cancer antigen-125 (CA-125)assessable disease according to Gynecologic Center Intergroup (GCIG)criteria. The following histological types are eligible:

-   -   Adenocarcinoma not otherwise specified.    -   Clear cell adenocarcinoma.    -   Endometrioid adenocarcinoma.    -   Malignant Brenner's tumor.    -   Mixed epithelial carcinoma including malignant mixed Müllerian        tumors.    -   Mucinous adenocarcinoma.    -   Serous adenocarcinoma.    -   Transitional cell carcinoma.    -   Undifferentiated carcinoma.

5. Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2.

6. LVEF greater than or equal to 55%.

Pertuzumab Dosage and Administration

Pertuzumab and Pertuzumab-Placebo Will be Administered as an IntravenousInfusion on Day 1 of the first treatment cycle as a loading dose of 840,followed by 420 mg on Day 1 of each subsequent 3-weekly cycle. Theinitial infusion of Pertuzumab/Pertuzumab-placebo will be administeredover 60 minutes followed by a 60-minute observational period in a seatedposition if the infusion is well tolerated, subsequent infusions may begiven over 30 minutes, followed by a 30-minute observational period,after which the chemotherapy agent will be administered. Pre-medicationshould be implemented according to local practices and the chosenchemotherapy.

Topotecan Dosage and Administration

Topotecan should be administered 1.25 mg/m² as a 30-minute intravenousinfusion daily on Days 1-5 every 3 weeks, as per the directions in thesummary of product characteristics.

Paclitaxel Dosage and Administration

Paclitaxel should be administered 80 mg/m2 as a 1-hour i.v. infusion onDays 1, 8, 15 and 22. Pharmacists should follow the summary of productcharacteristics for information regarding the preparation andadministration of the 80 mg/m2 dose.

Gemcitabine Dosage and Administration

Gemcitabine (Part 2 of the study only) should be administered 1000 mg/m2as a 30-minute intravenous infusion on Days 1 and 8 every 3 weeks as perthe directions described in the summary of product characteristics.

HER3 mRNA Expression

Patients will be asked to specifically consent to the collection andtesting of primary tumor tissue samples to assess HER3 mRNA level,including mRNA and protein levels of other HER family receptors e.g.HER2, before they provide consent to participate in the trial. Onlypatients who have tumors expressing low levels of HER3 mRNA will beeligible to participate in the trial.

During the initial screening for HER3 mRNA levels, other receptors ofthe HER family (e.g. EGFR, HER2, or HER4) will be assessed at the mRNAlevel and/or protein level in parallel to the HER3 assessment, in orderto obtain a more complete picture of the status of HER family receptorsby mRNA level.

The cut-off defined for study eligibility is defined as a concentrationratio of ≤2.81 as assessed by qRT-PCR on a COBAS z480® instrument usingthe “COBAS® HER2 & HER3 (qRT-PCR) mRNA expression assay” provided byRoche Molecular Diagnostics. The rationale for cut off definition isbased on a cut off modeling in previous studies as well as on atransformation function that had to be introduced since the assay wasswitched to a new instrument, the COBAS z480®. It is anticipated that40-50% of screened patients will have HER3 mRNA levels below the cutoffof 2.81 and that 30% of patients expressing low levels of HER3 mRNA willbe ineligible for enrollment owing to other inclusion/exclusioncriteria.

Submission of a formalin-fixed, paraffin-embedded tumor specimen of theprimary tumor from the original surgery will be required for allpatients prior to screening; cytology specimens are not acceptablereplacements. Patients will be assessed for HER3 mRNA expression level,as well as mRNA expression and protein expression levels of other HERfamily receptors by the use of a qRT-PCR assay and IHC. Such assessmentof HER receptor mRNA/protein expression will occur after obtaining thepatient's informed consent at any time after the primary surgery andprior to screening.

It is anticipated that Pertuzumab in combination with topotecan orpaclitaxel will be safe and effective in patients with epithelialovarian, primary peritoneal, or fallopian tube cancer.

In addition, it is anticipated that Pertuzumab plus chemotherapy(topotecan, paclitaxel, or gemcitabine) will be superior to placebo pluschemotherapy in patients with epithelial ovarian, primary peritoneal, orfallopian tube cancer where efficacy is measured by PFS.

1-42. (canceled)
 43. A method of treating early-stage HER2-positive breast cancer in a patient comprising administering Pertuzumab, Trastuzumab, and carboplatin-based chemotherapy to the patient with the early-stage breast cancer.
 44. The method of claim 43, wherein the carboplatin-based chemotherapy comprises Docetaxel and Carboplatin.
 45. The method of claim 43, wherein the Pertuzumab is administered concurrently with the carboplatin-based chemotherapy.
 46. The method of claim 43, wherein Pertuzumab administration does not increase cardiac toxicity relative to administration without Pertuzumab.
 47. The method of claim 43, which comprises neoadjuvant or adjuvant therapy.
 48. The method of claim 43, wherein the early-stage breast cancer is >2 cm in diameter.
 49. The method of claim 43, wherein the early-stage breast cancer has not spread beyond the breast or the axillary lymph nodes.
 50. The method of claim 43, wherein the early-stage breast cancer is locally advanced or inflammatory breast cancer.
 51. The method of claim 43, wherein the patient has no metastases.
 52. The method of claim 43, wherein the early-stage breast cancer is estrogen receptor (ER) and progesterone receptor (PR) negative.
 53. The method of claim 43, wherein the neoadjuvant therapy results in a pathological complete response (pCR) in the patient.
 54. The method of claim 53, wherein the neoadjuvant treatment comprises Docetaxel/Carboplatin/Trastuzumab (TCH)+Pertuzumab (P) for 6 cycles.
 55. The method of claim 54, wherein the neoadjuvant treatment results in pathological complete response (pCR) rate greater than pCR rate with Fluorouracil/Epirubicin/Cyclophosphamide (FEC), Trastuzumab (H), and Pertuzumab (P) for 3 cycles followed by 3 cycles of Docetaxel (T), H, and P (FEC+H+P×3→T+P+H×3) and than pCR rate with FEC for 3 cycles followed by 3 cycles of T, H, and P (FEC×3→T+P+H×3).
 56. A method of treating a HER2-positive cancer patient comprising administering to the patient an initial dose of 840 mg of Pertuzumab followed every 3 weeks thereafter by a dose of 420 mg of Pertuzumab, and further comprising re-administering an 840 mg dose of Pertuzumab to the patient if the time between two sequential 420 mg doses is 6 weeks or more.
 57. The method of claim 56, further comprising administering 420 mg of Pertuzumab every 3 weeks after the re-administered 840 mg dose.
 58. The method of claim 56, wherein the cancer patient has HER2-positive breast cancer.
 59. A method of combining two HER2 antibodies to treat HER2-positive breast cancer without increasing cardiac toxicity in a HER2-positive breast cancer patient with a left ventricular ejection fraction (LVEF) of ≤50% pre-treatment, comprising administering Pertuzumab, Trastuzumab, and chemotherapy to the patient, and measuring cardiac toxicity in the patient for incidence of symptomatic left ventricular systolic dysfunction (LVSD), congestive heart failure (CHF), or decrease in left ventricular ejection fraction (LVEF), wherein the cardiac toxicity is not increased compared to cardiac toxicity in a patient treated with Trastuzumab and the chemotherapy.
 60. The method of claim 59, wherein the breast cancer is metastatic breast cancer.
 61. The method of claim 59, wherein the breast cancer is metastatic or locally recurrent, unresectable breast cancer, or de novo Stage IV disease.
 62. The method of claim 59, wherein the chemotherapy comprises taxane.
 63. The method of claim 62, wherein the taxane is Docetaxel.
 64. The method of claim 59, wherein the cardiac toxicity is LVSD.
 65. The method of claim 59, wherein the cardiac toxicity is CHF.
 66. The method of claim 59, wherein the cardiac toxicity is decrease in LVEF.
 67. The method of claim 59, wherein the patient has not received chemotherapy or biologic therapy for their metastatic disease prior to treatment.
 68. A method of treating a patient with HER2-positive breast cancer comprising administering Pertuzumab, Trastuzumab, and chemotherapy to the patient, and measuring left ventricular ejection fraction (LVEF) in the patient and withholding treatment with pertuzumab and trasutuzmab for at least 3 weeks if the patient has a drop in LVEF to less than 40%, or a LVEF of 40% to 45% with a 10% or greater absolute decrease below pre-treatment value.
 69. The method of claim 68, further comprising resuming treatment with pertuzumab if the LVEF has recovered to greater than 45%, or to 40% to 45% associated with less than a 10% absolute decrease below pretreatment value. 